Cell And Enzyme Compositions For Modulating Bile Acids, Cholesterol and Triglycerides

ABSTRACT

The invention relates to immobilized or encapsulated enzyme and/or cells to lower bile acids and cholesterol. The invention also relates to methods of quantitatively measuring bile acids. The invention provides a composition for decreasing the amount of a target compound in the gastrointestinal tract of an animal, comprising: a) a biologically active agent which decreases the amount of the target compound; b) a retainer for retaining the biologically active agent by contacting the agent to limit movement of the agent; and c) a carrier.

FIELD OF THE INVENTION

The invention relates to immobilized or encapsulated enzyme and/or cellsto modulate bile acids, cholesterol and triglyceride levels in asubject. The invention also relates to methods of quantitativelymeasuring bile acids and triglycerides.

BACKGROUND OF THE INVENTION

Bile acids are important physiological agents that are required for thedisposal of cholesterol and the absorption of dietary lipids and lipidsoluble vitamins. Bile salts are the water-soluble end products ofcholesterol, and are synthesized de novo in the liver. During normalenterohepatic circulation (EHC), the average bile salt pool is secretedinto the duodenum twice during each meal, or an average of 6-8 times perday for the purpose of forming mixed micelles with the products of lipiddigestion. During intestinal transit, most of the secreted bile salt isabsorbed in the terminal ileum and is returned to the liver via theportal vein. The bile salt pool is replenished by hepatic synthesis ofnew bile from serum cholesterol. It has been shown that upon surgical,pharmacological or pathological interruption of the EHC, bile saltsynthesis is increased up to 15-fold, leading to an increased demand forcholesterol in the liver. Therefore, various studies have been reportedsuggesting possible oral bacterial preparations for reducing serumcholesterol. Though effective, these methods still have severallimitations. For example, a normal daily intake of 250 ml of yogurtwould only correspond to 500 milligram of cell dry weight (CDW) ofbacteria, and of those bacteria ingested only 1% would survive gastrictransit limiting the overall therapeutic effect. There are also somepractical concerns regarding the production, cost, and storage of such aproduct (De Smet et al., 1998). Further, oral administration of livebacterial cells can pose problems. For example, when given orally, largeamounts of live bacterial cells can stimulate host immune response, theycan be retained in the intestine, and repeated large doses could resultin their replacing the normal intestinal flora (De Boever andVerstraete, 1999; Christiaens et al., 1992). In addition, risk ofsystemic infections, deleterious metabolic activities, adjuvantside-effects, immuno-modulation and risk of gene transfer has limitedtheir use (De Boever and Verstraete, 1999; Christiaens et al., 1992).Metabolic activities and immuno-modulation, have limited its clinicaluse (De Boever et al., 2000).

Although bile acids are important to normal human physiology, bile acidscan be cytotoxic agents when produced in pathologically highconcentrations. As well, when ileal transport of bile acids is defectivedue to a congenital defect, resection of the ileum, or disease, elevatedintraluminal concentrations of bile acids can induce the secretion ofelectrolytes and water causing diarrhea and dehydration. Therefore,various studies suggested methods for removing bile acids by eitherdirectly preventing the reabsorption of bile acids or by removing bileacids using chemical binders such as bile acid sequestrants (BAS). Thesemethods have several limitations. For example, common BAS Cholestyramineresin (Locholest, Questran), Colesevelam (Welchol), and Colestipol(Colestid) are well documented to exhibit major adverse effects such asnausea, bloating, constipation, and flatulence (Christiaens et al.,1992).

Current treatments for elevated blood cholesterol include dietarymanagement, regular exercise, and drug therapy with fibrates, bile acidsequestrants, and statins. Such therapies are often sub-optimal andcarry a risk for serious side effects. Dietary intervention, wherebylipid intake is restricted is generally the first line of treatment(Lichtenstein, 1998; Ornish and Denke, 1994; Ornish et al., 1998).Studies show that complete elimination of dietary cholesterol andlimiting fat content to less than ten percent of the daily caloricintake can effect a mere four percent regression of atheroscleroticplaques after five years when combined with stress management andaerobic exercise (Dunn-Emke et al., 2001). However, the combinedrestricted vegetarian diet (free of meat, fish, chicken, vegetable oilsand all dairy fat products) and aerobic approach, is unrealistic for allbut the most dedicated individuals. A variety of dietary supplements orspecific foods e.g. brans, psylliums, guar gum, lecithins, whey, redwines, fish oils and ginseng root extract have been reported to reducehigh blood cholesterol or its consequences. The mechanisms are variedand include cholesterol sequestering, chelating, entrapment andoxidation inhibition. Such regimens generally lower the bloodcholesterol level by ten percent or less. In addition, none of thesedietary interventions have been shown to arrest or cure atherosclerosisor other high blood cholesterol associated diseases.

Pharmacologic agents such as fibric acid derivatives (fibrates),nicotinic acid, bile acid sequestrants (BAS), estrogen replacementtherapy, and hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductaseinhibitors (statins) are also available for the treatment of highcholesterol. From among the agents listed above, the statins areconsidered to have the most potential for treatment. Currentlylovastatin, pravastatin, zocor, fluvastatin and atorvastatin are beingused for clinical lowering of cholesterol. Although effective atreducing cholesterol levels, they are nevertheless expensive (Attanasioet al., 2001; Hodgson and Cohen, 1999; Prosser et al. 2000; Reckless,1996). Some are known to have side effects and are associated. Naturallyoccurring bacteria can significantly lower serum cholesterol levels byhydrolyzing bile salts in the intestinal tract but only 1% of freebacteria ingested survive the GI transit. However, live bacterial cellscan cause a host immune response and can be retained in the intestinereplacing the natural intestinal flora (Taranto et al., 2000; Andersonand Gilliland, 1999; Chin et al., 2000). It has been shown that certainstrains of bacteria act directly on bile acids in the gastrointestinaltract and may be beneficial in reducing serum cholesterol levels in thisway (Taranto et al., 2000; Anderson and Gilliland, 1999; De Smet et al.1994). Control of cholesterol through oral live bacterial cell therapy,is based on the demonstration that naturally occurring bacteria such asLactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillusreuteri can significantly lower serum cholesterol levels (Taranto etal., 2000; Anderson and Gilliland, 1999; De Smet et al. 1994). Forexample, Lactobacillus reuteri was used to decrease the serumcholesterol in pigs through interaction of free bacteria with the host'sbile salt metabolism (De Smet et al., 1998). The underlying mechanismfor the reduction of serum cholesterol appears to be the capacity ofLactobacillus to hydrolyze bile salts in the intestinal tract (Andersonand Gilliland, 1999; De Smet et al. 1994). Elevated Bile Salt Hydrolase(BSH) activity leads to an increase in the loss of bile acids from theECH and to a greater demand for cholesterol by the liver (De Smet et al.1994) (FIG. 8). In the work of De Smet et al., the BSH activity of BSHoverproducing. Lactobacillus plantarum 80 (pCBHl) was shown to have aconsiderable cholesterol lowering capacity (De Smet et al. 1994). Thebile salt hydrolase enzyme, contained on the multicopy plasmid (pCBHl),carries out the deconjugation of bile salts through catalysis ofhydrolysis of the amide bond that conjugates bile acids to glycine ortaurine (Christiaens et al., 1992; De Smet et al. 1994) (FIG. 9).

While work in this field has been very promising, several limitingfactors to the oral administration of free bacteria have beenidentified. The therapeutic potential of free bacteria is hampered byinherent limitations in their use. For example, of those free bacteriaingested only 1% survive gastric transit limiting the overalltherapeutic effect (De Smet et al. 1994). Also, oral administration oflive bacterial cells can cause a host immune response, and can bedetrimentally retained in the intestine replacing the natural intestinalflora (Taranto et al., 2000; Chin et al., 2000; De Boever andVerstraete, 1999). Furthermore, there are some practical concernsregarding the production, cost, and storage of products containing freebacteria (De Boever and Verstraete, 1999). Thus, concerns of safety andpracticality have prevented the regular use of this promising therapy inclinical practice.

Other problematic diseases or disorders arise from disrupted lipidmetabolism. For example, steathorrea results from damage to the pancreasor bowel (eg. inflammation resulting from pancreatitis). The pancreas isthe gland that produces digestive enzymes to metabolize carbohydratesand lipids. The resulting condition, known as exocrine or pancreaticinsufficiency, leads to weight loss and very foul-smelling stools ordiarrhea. Chronic pancreatitis can lead to diabetes and pancreaticcalcification, a condition where small, hard deposits form in thepancreas. There is a need for new treatments that allow patients tofully digest food.

Encapsulation and immobilization patents include U.S. Pat. No.6,565,777, U.S. Pat. No. 6,346,262, U.S. Pat. No. 6,258,870, U.S. Pat.No. 6,264,941, U.S. Pat. No. 6,217,859, U.S. Pat. No. 5,766,907 and U.S.Pat. No. 5,175,093. Artificial cell microencapsulation is a techniqueused to encapsulate biologically active materials in specialized ultrathin semi-permeable polymer membranes (Chang and Prakash, 1997; Chang,1964). The polymer membrane protects encapsulated materials from harshexternal environments, while at the same time allowing for themetabolism of selected solutes capable of passing into and out of themicrocapsule. In this manner, the enclosed material is retained insideand separated from the external environment, making microencapsulationparticularly useful for biomedical and clinical applications (Lim andSun, 1980; Sefton et al, 2000; Chang, 1999). Studies show thatartificial cell microcapsules can be used for oral administration oflive genetically engineered cells that can be useful for therapeuticfunctions (Prakash and Chang, 2000; Prakash and Chang, 1996). Examplesof applications of microencapsulation of enzymes, cells and geneticallyengineered microorganisms are xanthine oxidase for Lesch-Nyhan disease;phenylalanine ammonia lyase for pheny, ketonuria and E. coli DH5 cellsfor lowering urea, ammonia and other metabolites (Chang and Prakash2001). Although the live cells remain immobilized inside themicrocapsules, microencapsulation does not appear to hinder their growthkinetics (Prakash and Chang, 1999). The microcapsules remain intactduring passage through the intestinal tract and are excreted intact withthe stool in about 24 hours. The cells are retained inside, and excretedwith, the intact microcapsules addressing many of the major safetyconcerns associated with the use of live bacterial cells for variousclinical applications. The membranes of the microcapsules are permeableto smaller molecules, and thus the cells inside the microcapsulesmetabolize small molecules found within the gut during passage throughthe intestine (Chang and Prakash, 1997; Prakash and Chang, 2000; Prakashand Chang, 1996; Prakash and Chang, 1999; Prakash and Chang, 1996a,Prakash and Chang, 1999a).

SUMMARY OF THE INVENTION

The invention relates to compositions and methods that are useful formodulating levels of a target compound, such as bile or triglycerides,in an animal. Typically, the compositions and methods modulate levels inthe gastrointestinal system of the animal. Adjusting the levels in thegastrointestinal system affects levels in serum and other fluids,tissues and excrement. The compositions and methods are useful forreducing bile and cholesterol levels in an animal to prevent or treat adisease or disorder characterized by increased bile and cholesterollevels (or a disease or disorder having increased bile or cholesterol asa risk factor, such as heart disease or cancer). The compositions andmethods are also useful for providing trigylceride-hydrolysis products,such as fatty acids and glycerol, to an animal in need thereof, forexample, an animal having pancreatitis or other disruptions of thepancreas or bowel.

The compositions are optionally orally administered or implanted in theanimal. The compositions act on target compound produced by the animalor consumed by the animal, for example target compound in food ornutritional supplements. The compositions are optionally pharmaceuticalcompositions, food compositions and/or nutraceutical compositions. Thecompositions optionally comprise:

i) a biologically active agent which modulates target compound levels inan animal, for example, by degrading target compound in an animal toreduce target compound levels. The agent is optionally an enzyme formodulating lipid or bile metabolism, such as BSH, for deconjugating bileacids to form target-degradation compounds. This has the effect ofreducing bile acid levels. The agent is also optionally a lipase, whichbreaks down lipids, such as triglycerides and their esters, to formtarget-degradation compounds such as fatty acids. The agent alsooptionally comprises a cell, such as a bacterial cell, expressing theenzyme;

ii) a retainer for retaining the biologically active agent, for exampleby immobilizing it on a surface and/or encapsulating it. This has theeffect of isolating the agent and reducing its movement. The retaineroptionally comprises a capsule, such as a capsule comprising asemi-permeable membrane, and/or a support, such as a polymer structure.The retainer is optionally a retainer means for retaining the agent; and

iii) a carrier. The carrier is optionally a pharmaceutically acceptablecarrier, such as saline solution.

In one embodiment, the compositions further comprise a collector forcollecting a target-degradation compound formed as a result of theagent's reaction with the target compound. Collection permits thetarget-degradation compound to be either excreted by the animal orabsorbed by the animal's gastrointestinal system.

The invention also includes methods comprising contacting a biologicallyactive agent (for example, a composition of the invention) with a targetcompound in an animal to, for example, degrade target compound in ananimal to reduce target compound levels. The methods optionally modulatelipid or bile metabolism, with an agent such as BSH, for deconjugatingbile acids to form target-degradation compounds. This has the effect ofreducing bile acid levels. The methods optionally use a lipase, whichbreaks down lipids, such as triglycerides and their esters, to formtarget-degradation compounds such as fatty acids. The methods optionallyuse a cell, such as a bacterial cell, expressing the enzyme. The methodsoptionally involve oral administration or implantation in the animal. Inthe methods, the biologically active agent is optionally retained in aretainer, for example immobilized on a surface and/or encapsulated. Thishas the effect of isolating the agent and reducing its movement in themethods. The methods optionally further comprise collecting atarget-degradation compound formed as a result of the agent's reactionwith the target compound. In one embodiment, a bile acid is deconjugatedand then, its by-product, DCA, is captured, for example by precipitationand collection in a capsule, where it is held until it is excreted.

In one embodiment of the invention, the present inventors have shownthat immobilized or encapsulated genetically engineered cells, such asLactobacillus plantarum 80 cells expressing BSH, are a biologicallyactive agent that efficiently hydrolyzes bile acids and that are usefulin the deconjugation of human bile acids.

Another embodiment of the invention relates to cells, for example,immobilized or encapsulated genetically engineered cells, such asLactobacillus cells expressing lipase, as a biologically active agentthat efficiently hydrolyzes lipids and that are useful in the hydrolysisof human lipids.

Accordingly, in an embodiment, the present invention provides acomposition of immobilized or encapsulated cells, such as bacteria,and/or enzyme for lowering bile acids and/or cholesterol.

In another embodiment, the present invention provides a compositioncomprising of at least one immobilized and biologically active agent inan amount sufficient to degrade bile acids or lipids and a carrier. Thebiologically active agent is optionally any cell expressing or capableof expressing a bile acid degrading enzyme or lipid-degrading enzyme,anaerobic bacteria expressing or capable of expressing a bile aciddegrading enzyme or a lipid-degrading enzyme, a bile acid degradingenzyme-containing cell extract, a lipid-degrading enzyme-containing cellextract, a bile acid degrading enzyme itself or a lipid-degrading enzymeitself. Cells or bacteria are optionally genetically engineered. Thebacteria is optionally Lactobacillus such as, Lactobacillus plantarum,Lactobacillus reuteri or a combination thereof. Bile acid degradingenzymes include BSH. BSH is optionally lactobacillus plantarum BSH.Lipid degrading enzymes include lipase, such as mammalian or bacteriallipase.

The immobilized biologically active agent is usefully encapsulated ormicroencapsulated.

In one embodiment, the carrier is intended for oral administration andis optionally in the form of a nutraceutical or functional food product.

The invention includes the use compositions of the invention for use inmedicine (eg. as a pharmaceutical substance). The invention alsoincludes the use of compositions of the invention for the manufacture ofa medicament effective against diseases and disorders recited in thisapplication. Unwanted intraluminal bile acids in the gastrointestinalsystem are associated with bowel diseases. Accordingly, the presentinvention provides a method for lowering of intraluminal bile acid ofpatients suffering from a bowel disease, which comprises ofadministering a bile acid lowering amount of a composition of thepresent invention.

Naturally occurring bacteria can significantly lower serum cholesterollevels by hydrolyzing bile salts in the intestinal tract. Accordingly,the present invention provides a method for lowering of serumcholesterol of patients, which comprises administering a bile acidlowering amount of a composition of the present invention.

The composition for lowering of intraluminal bile acids or serumcholesterol may be administered singly or in combination with othercholesterol lowering therapeutics.

In another embodiment, the present invention provides a method forlowering of serum cholesterol and/or total body cholesterol of animalsfor the purpose of producing animal products of reduced cholesterolcontent, which comprises administering a bile acid lowering amount of acomposition of the present invention.

Colon cancer has been linked to diet and the proposed mechanism is thata high fat diet leads to an increased secretion of primary bile saltsinto the small intestine where the indigenous microflora deconjugatesthe primary bile acids. Accordingly, the present invention provides amethod for preventive therapy of colon cancer in a patient, whichcomprises administering a bile acid lowering amount of a composition ofthe present invention.

Urinary levels of sulfated bile acids are known to be significantlyelevated in liver disease and hepatobiliary disease. Accordingly, thepresent invention provides an in vitro diagnostic tool for liver andhepatobiliary diseases and disorders in an animal (eg. a patient), whichcomprises

a) support;

b) a biologically active agent immobilized onto said support;

wherein the immobilized agent allows detection and measurement of bileacid degradation when contacted with a biological sample. Thebiologically active agent is optionally any cell expressing or capableof expressing a bile acid degrading enzyme, anaerobic bacteriaexpressing or capable of expressing a bile acid degrading enzyme, a bileacid degrading enzyme-containing cell extract or a bile acid degradingenzyme. Bile acid degrading enzymes include BSH. BSH is optionallylactobacillus plantarum BSH. The diagnostic tool is readily adapted tomeasure lipids and diagnose a disease or disorder characterized byimproper/inadequate lipid hydrolysis in an animal.

The present invention also provides a method for quantitativelymeasuring bile acids. In an embodiment, the present invention providesan in vitro method for measuring bile acid which comprises

(a) contacting a biological sample with the tool of the presentinvention

(b) contacting a control sample with the tool of the present invention

(c) comparing the amount of degradation of bile acid in (a) and (b)

wherein a higher level of degradation product compared to control levelis indicative of a liver or hepatobiliary disease. The diagnostic toolis readily adapted to quantitatively measure lipids in an animal.

The present invention provides a composition for decreasing the amountof a target compound in the gastrointestinal tract of an animal,comprising:

-   -   i) a biologically active agent which decreases the amount of the        target compound;    -   ii) a retainer for retaining the biologically active agent by        contacting the agent to limit movement of the agent;    -   iii) a carrier.

In one embodiment, the retainer limits agent movement by a retainersurface immobilizing the agent and/or by the retainer encapsulating theagent. In a further embodiment, the retainer encapsulates the agent andreduces exposure of the biologically active agent to antibodies andpermits exposure of the biologically active agent to nutrients. Theretainer optionally comprises a semi-permeable membrane. Thesemi-permeable membrane also optionally comprises a MWCO of about 3000 Dto 950,000 D In one embodiment, the retainer comprises a polymer beadand the agent is immobilized on the bead.

The target compound optionally comprises bile acid or triglyceride andthe amount of the target compound is decreased by degrading the targetcompound to at least one target-degradation compound. In one embodiment,the target compound comprises bile acid and the target-degradationcompound comprises DCA. In another embodiment, the target compoundcomprises triglyceride and the target-degradation compound comprisesfatty acid.

The invention further provides for a composition comprising a collectorfor collecting the target-degradation compound and permitting the animalto excrete or absorb the target-degradation compound from thegastrointestinal tract of the animal. In one embodiment, the retainercomprises the collector. Optionally, the target-degradation compound forcollection comprises a DCA precipitate or a fatty acid.

In another embodiment, the biologically active agent is selected fromthe group consisting of a cell expressing a bile acid degrading enzyme,anaerobic bacteria expressing a bile acid degrading enzyme, a bile aciddegrading enzyme-containing cell extract, or a bile degrading enzyme.

In a further embodiment, the biologically active agent is selected fromthe group consisting of a cell expressing a triglyceride degradingenzyme, anaerobic bacteria expressing a triglyceride degrading enzyme, atriglyceride degrading enzyme-containing cell extract, or a triglyceridedegrading enzyme.

The cells optionally comprise a human cell, a fungal cell or a bacterialcell. In a further embodiment the bacteria or cell is geneticallyengineered. The bacteria optionally comprises at least one ofLactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium bifidum,Lactobacillus acidophilus, and Clostridium perfringens. Alternatively,the bacteria comprises a combination of Lactobacillus plantarum, andLactobacillus reuteri. In one embodiment, the Lactobacillus plantarumcomprises Lactobacillus plantarum 80.

In a further embodiment, the bile acid degrading enzyme comprises BSH.BSH optionally has a nucleotide sequence as shown in one of SEQ. ID. NO.1, 5, 7 or 9 and an amino acid sequence as shown in one of SEQ. ID. NO.2, 6, 8 or 10.

In another embodiment, the triglyceride bile acid degrading enzymecomprises lipase. The lipase optionally has a nucleotide sequence asshown in SEQ. ID. NO. 3 and an amino acid sequence as shown in SEQ. ID.NO. 4.

In one embodiment, the biologically active agent is encapsulated ormicroencapsulated in a membrane made of alginate-polylysine-alginate(APA). Alternatively, the biologically active agent is encapsulated ormicroencapsulated in a membrane made ofAlginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP), andAlginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA) membranes

In another embodiment, the carrier comprises an orally administerablecarrier. In a further embodiment, the carrier comprises a nutraceuticalor functional food product. Alternatively, the carrier comprises animplantable device.

In one embodiment, the composition comprises a pharmaceuticalcomposition and the carrier comprises a pharmaceutically acceptablecarrier.

The invention also provides a method for lowering of intraluminal bileacid of animals suffering from defective ileal transport of bile acidsdue to a congenital defect, resection of the ileum or a bowel disease ordisorder, which comprises administering to the animal a bile acidlowering amount of a composition of the invention.

The invention further provides a method for lowering of intraluminalbile acid or patients, comprising administering to the animal a capsuleor immobilized agent comprising:

(a) a first bacteria that deconjugates bile salts and

(b) a second bacteria that precipitates and binds the deconjugated bilesalts.

In one embodiment, the first bacteria is L. plantarum and the secondbacteria is L. reuteri.

The invention also provides for a method for lowering serum cholesterolof an animal, comprising administering to the animal a bile acidlowering amount of a composition of the invention.

The invention provides for a method for lowering serum cholesteroland/or total body cholesterol of animals for the purpose of producinganimal products of reduced cholesterol content, comprising administeringto the animal a bile acid lowering amount of a composition of theinvention. In a further embodiment, the composition is administered incombination with another cholesterol lowering therapeutic. The anothercholesterol lowering therapeutic is optionally selected from the groupconsisting of BAS Cholestyramine resin, Colesevelam, Colestipol, statin,probiotic formulation containing other live bacterial cells andneutraceuticals, and natural cholesterol lowering products. In oneembodiment, the statin is selected from the group consisting oflovastatin, pravastatin, zocor, fluvastatin, and atorvastatin

The invention further provides for a method for preventing or treatingcolon cancer in an animal, which comprises administering to the animal abile acid lowering amount of a composition of the invention.

In an embodiment, the invention provides an in vitro diagnostic tool fordetecting liver or hepatobiliary disease in a patient, which comprises

-   -   a) a support;    -   b) a biologically active agent immobilized onto the support;        wherein the immobilized agent allows detection and/or        measurement of bile acid degradation when contacted with a        biological sample.

In one embodiment, the biological sample comprises urine, blood, fecesor vomit. The detection is optionally based on a colour indicatorwherein a change in colour of the indicator in contact with thebiological sample compared to the colour of a control is indicative ofbile acid degradation and reduced or increased bile acid in an animalcompared to normal animal bile acid degradation is indicative of liveror hepatobiliary disease. The biologically active agent is optionallyselected from the group consisting of a cell expressing a bile aciddegrading enzyme, anaerobic bacteria expressing a bile acid degradingenzyme, a bile acid degrading enzyme-containing cell extract, or a biledegrading enzyme. The cell is optionally a human cell, a fungal cell ora bacterial cell. The bacteria or cell is also optionally geneticallyengineered. In one embodiment, the bacteria comprises at least one ofLactobacillus plantarum, Lactobacillus reuteri, Bifidobaterium bifidum,Lactobacillus acidophilus, and Clostridium perfringenes. Alternatively,the bacteria comprises a combination of Lactobacillus plantarum, andLactobacillus reuteri. The Lactobacillus plantarum optionally comprisesLactobacillus plantarum 80 (pCBHl).

In another embodiment, the bile acid degrading enzyme of the in vitrodiagnostic tool comprises BSH. The BSH optionally has a nucleotidesequence as shown in SEQ. ID. NO. 1, 5, 7, or 9 and an amino acidsequence as shown in SEQ. ID. NO. 2, 6, 8, or 10.

In a further embodiment, the immobilized biologically active agent ofthe in vitro diagnostic tool is encapsulated or microencapsulated. Thebiologically active agent is optionally encapsulated ormicroencapsulated in a membrane comprising alginate-polylysine-alginate(APA). Alternatively, the biologically active agent is encapsulated ormicroencapsulated in a membrane comprisingAlginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP), andAlginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA) membranes

The invention also provides for an in vitro method for measuring bileacid comprising

-   -   a) contacting a biological sample with a tool of the invention    -   b) contacting a control sample with a tool of the invention    -   c) comparing the amount of degradation of bile acid in (a) and        (b)        wherein a higher level of degraded bile acid product in (a)        than (b) is indicative of a liver or hepatobiliary disease.

The invention provides a method for lowering triglycerides, whichcomprises administering a triglyceride lowering amount of a compositionof the invention.

The invention also provides a method for lowering total body fat ofanimals for the purpose of producing animal products of reduced fatcontent, comprising administering a triglyceride lowering amount of acomposition of the invention.

The invention further provides a method for preventing or treatingsteathorrea in a patient, which comprises administering a triglyceridelowering amount of a composition of the invention.

In one embodiment, the invention provides an in vitro diagnostic toolfor detecting steathorrea in an animal, which comprises

-   -   a) a support;    -   b) a biologically active agent immobilized onto the support;        wherein the immobilized agent allows detection and/or        measurement of triglyceride degradation when contacted with a        biological sample.

In one embodiment, the biological sample comprises urine, blood, fecesor vomit. The detection is optionally based on a colour indicatorwherein a change in colour of the indicator in contact with thebiological sample compared to the colour of a control is indicative oftriglyceride degradation in the animal compared to normal animaltriglyceride degradation is indicative of steathorrea. The biologicallyactive agent is optionally selected from the group consisting of a cellexpressing a triglyceride degrading enzyme, anaerobic bacteriaexpressing a triglyceride degrading enzyme, a triglyceride degradingenzyme-containing cell extract, or a triglyceride degrading enzyme. Thecell is optionally a human cell, a fungal cell or a bacterial cell. Thebacteria or cell is also optionally genetically engineered. In oneembodiment, the bacteria comprises at least one of Lactobacillusplantarum, Lactobacillus reuteri, Bifidobaterium bifidum, Lactobacillusacidophilus, and Clostridium perfringenes. Alternatively, the bacteriacomprises a combination of Lactobacillus plantarum, and Lactobacillusreuteri. The Lactobacillus plantarum optionally comprises Lactobacillusplantarum 80 (pCBHl).

In another embodiment, the triglyceride degrading enzyme of the in vitrodiagnostic tool comprises lipase. The lipase optionally has a nucleotidesequence as shown in SEQ. ID. NO. 3 and an amino acid sequence as shownin SEQ. ID. NO. 4.

In a further embodiment, the immobilized biologically active agent ofthe in vitro diagnostic tool is encapsulated or microencapsulated. Thebiologically active agent is optionally encapsulated ormicroencapsulated in a membrane comprising alginate-polylysine-alginate(APA). Alternatively, the biologically active agent is encapsulated ormicroencapsulated in a membrane comprisingAlginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP), andAlginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA) membranes

The invention further provides for an in vitro method for measuringtriglyceride comprising

a) contacting a biological sample with the tool of any one of claims 61to 74

b) contacting a control sample with the tool of any one of claims 61 to74

c) comparing the amount of degradation of triglyceride in (a) and (b)

wherein a higher level of degradation product in (a) than (b) isindicative of high triglyceride fat content.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described in relation to the drawingsin which:

FIG. 1 illustrates a photomicrograph of alginate beads containingimmobilized Lactobacillus plantarum 80 (pCBHl) cells at 43.75×magnification (A) and at 175× magnifications (B);

FIG. 2 illustrates a photomicrograph of Lactobacillus plantarum 80(pCBHl) microcapsules at 77× magnification (A) and at 112×magnifications (B);

FIG. 3 illustrates HPLC calibration curves for GDCA and TDCAmeasurements;

FIG. 4 illustrates overlaid HPLC chromatograms of bile acids in reactionmedia over time (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). Decreasing peakareas of TDCA and GDCA indicate BSH activity of immobilizedLactobacillus plantarum 80 (pCBHl);

FIG. 5 illustrates overlaid HPLC chromatograms of bile acids in reactionmedia over time (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). Decreasing peakareas of TDCA and GDCA indicate BSH activity of Lactobacillus plantarum80 (pCBHl) microcapsules;

FIG. 6 illustrates BSH activity and GDCA and TDCA depletion efficiencyof immobilized Lactobacillus plantarum 80 (pCBHl) in an in-vitroexperiment. The concentration of GDCA and TDCA bile acids are shown overtime. The experiment was performed in triplicate: error bars indicatestandard deviations;

FIG. 7 illustrates BSH activity and GDCA and TDCA depleting efficiencyof Lactobacillus plantarum 80 (pCBHl) microcapsules in an in-vitroexperiment. The concentration of GDCA and TDCA bile acids are shown overtime. The experiment was performed in triplicate: error bars indicatestandard deviations;

FIG. 8 illustrates the Enterohepatic circulation of bile (EHC);

FIG. 9 illustrates hydrolysis of conjugated bile salts by the Bile SaltHydrolase (BSH) enzyme overproduced by genetically engineeredLactobacillus plantarum 80 (pCBHl). R indicates the amino acid glycineor taurine. RDCA: glyco- or tauro-deoxycholic acid, DCA: deoxycholicacid;

FIG. 10 illustrates (A) overlaid HPLC chromatograms of samples (0 h, 1h, 2 h, 3 h, 4 h, 5 h, 6 h) from experiment in which microencapsulatedLP80 (pCBHl) was used to deconjugate 10 mM GDCA and 5 mM TCDA. (B)Overlaid HPLC chromatograms of samples (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6h) from experiment in which immobilized LP80 (pCBHl) was used todeconjugate 10 mM GDCA and 5 mM TCDA. (C) Graphical representation (A),(D) Graphical representation of (B).

FIG. 11 illustrates APA microcapsules containing genetically engineeredLactobacillus plantarum 80 (pCBHl) and L. reuteri. BSH is overproducedby LP80 (pCBHl) cells and hydrolyzes available conjugated bile acids. L.reuteri precipitates and binds the produced deconjugated bile acidsmaking them unable to leave the microcapsule and thus less bioavailable;

FIG. 12 illustrates APA microcapsules containing genetically engineeredLactobacillus plantarum 80 (pCBHl) and L. reuteri. BSH is overproducedby LP80 (pCBHl) cells and hydrolyzes available conjugated bile acids. L.reuteri precipitates and binds the produced deconjugated bile acidsmaking them unable to leave the microcapsule and thus less bioavailable;

FIG. 13 illustrates diagnostic strip for determination of liver functionand detection of hepatobilary diseases through detection of conjugatedbile acids in urine. APA microcapsules containing genetically engineeredLP80 (pCBHl) and L. reuteri, as well as a colored detector molecule foreither the deconjugated bile acid or released amino acid, adhered to thefunctional end of a diagnostic strip. BSH is overproduced by LP80(pCBHl) cells and hydrolyzes available conjugated bile acids. L. reuteriprecipitates and binds the produced deconjugated bile acids. A coloreddetector molecule would react with either the deconjugated bile acid orreleased amino acid groups and produce a discernable change in color.(BA) is bile acid. (DBA) is deconjugated bile acid; and

FIG. 14 illustrates the hollow fiber membrane of a bioartificial liver(BAL) is impregnated with hepatocytes and APA microcapsules containinggenetically engineered Lactobacillus plantarum 80 (pCBHl) and L.reuteri. BSH is overproduced by LP80 (pCBHl) cells and hydrolyzesavailable conjugated bile acids. L. reuteri precipitates and binds theproduced deconjugated bile acids making them unable to leave themicrocapsule.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods that are useful formodulating levels of a target compound, such as bile or triglycerides,in an animal. Typically, the compositions and methods modulate levels inthe gastrointestinal system of the animal. Adjusting the levels in thegastrointestinal system affects levels in serum and other fluids,tissues and excrement. The compositions and methods are useful forreducing bile and cholesterol levels in an animal to prevent or treat adisease or disorder characterized by increased bile and cholesterollevels or a disease or disorder having increased bile or cholesterol asa risk factor, such as heart disease or cancer. The compositions andmethods are also useful for providing trigylceride-hydrolysis products,such as fatty acids and glycerol, to an animal in need thereof, forexample, an animal having pancreatitis or other disruptions of thepancreas or bowel.

The compositions act on target compound produced by the animal orconsumed by the animal, for example target compound in food ornutritional supplements. The compositions optionally comprise:

i) a biologically active agent which modulates target compound levels inan animal, for example, by degrading target compound in an animal toreduce target compound levels. The agent is optionally an enzyme formodulating lipid or bile metabolism, such as BSH, for deconjugating bileacids to form target-degradation compounds. This has the effect ofreducing bile acid levels. The agent is also optionally a lipase, whichbreaks down lipids, such as triglycerides and their esters, to formtarget-degradation compounds such as fatty acids. The agent alsooptionally comprises a cell, such as a bacterial cell, expressing theenzyme;

ii) a retainer for retaining the biologically active agent, for exampleby immobilizing it on a surface and/or encapsulating it. This has theeffect of isolating the agent and reducing its movement. The retaineroptionally comprises a capsule, such as a capsule comprising asemi-permeable membrane, and/or a support, such as a polymer structure.The retainer is optionally a retainer means for retaining the agent; and

iii) a carrier.

In one embodiment, the compositions further comprise a collector forcollecting a target-degradation compound formed as a result of theagent's reaction with the target compound. Collection permits thetarget-degradation compound to be either excreted by the animal orabsorbed by the animal's gastrointestinal system. The collectoroptionally contains (holds), binds, metabolizes or precipitates thetarget-degradation compound. The collector makes the target-degradationcompound less bioavailable. The collector is optionally a capsule orpolymer surface. The collector is also optionally a chemical associatedwith the capsule or polymer surface, for example, forming part of acapsule membrane or surface. The chemical may also be located in a spacedefined by the membrane or polymer surface. Alternatively, thetarget-degradation compound is physically contained (held) in a spacedefined by the membrane or polymer surface. The collector is optionallya collection means for collecting the target-degradation compound. Inone embodiment, the retainer itself performs the collector function bycollecting a target-degradation compound.

The invention also includes methods comprising contacting a biologicallyactive agent (for example, a composition of the invention) with a targetcompound in an animal to, for example, degrade target compound in ananimal to reduce target compound levels. The methods optionally modulatelipid or bile metabolism, with an agent such as BSH, for deconjugatingbile acids to form target-degradation compounds. This has the effect ofreducing bile acid levels. The methods optionally further comprisecollecting a target-degradation compound formed as a result of theagent's reaction with the target compound. In one embodiment, a bileacid is deconjugated and then, its by-product, DCA, is captured, forexample by precipitation and collection in a capsule, where it is helduntil it is excreted.

The invention is described in additional detail below.

The present inventors have demonstrated that immobilized or encapsulatedcells or enzymes efficiently hydrolyze bile acids and are useful in thedeconjugation of bile acids in animals, such as humans. Immobilized orencapsulated cells or enzymes also efficiently hydrolyze lipids inanimals, such as humans. An example of a suitable cell is geneticallyengineered Lactobacillus plantarum 80 (pCBHl) expressing BSH. Resultsshow that immobilized LP80 (pCBHl) is able to effectively break down theconjugated bile acids glycodeoxycholic acid (GDCA) and taurodeoxycholicacid (TDCA) with bile salt hydrolase (BSH) activities of 0.17 and 0.07μmol DCA/mg CDW/h respectively. In addition, the immobilized orencapsulated cells collect the DCA so that it is excreted. This is avery useful aspect because DCA is toxic and causes diseases, such ascancer. Immobilized live engineered cells are a good agent for thedeconjugation of bile acids and provide an effective therapy to lowerpathologically high levels of bile acids for prophylaxis or treatment ofdiseases and disorders caused by high levels of bile acids and/orcholesterol. Immobilized, live engineered cells are also a good agentfor the hydrolysis of lipids and provide an effective therapy where thesubject is unable to hydrolyze adequate amounts of lipids.

Accordingly, in an embodiment, the present invention provides acomposition of immobilized or encapsulated cells, such as bacteria, orenzyme for lowering bile acids and/or cholesterol. The phrase “bile acidlowering amount” as used herein means an amount effective, at dosagesand for periods of time necessary to achieve the desired results (e.g.degradation of bile acids and/or lowering of cholesterol). Effectiveamounts may vary according to factors such as the disease state, age,sex, weight of the animal. Dosage regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionately reduced asindicated by the exigencies of the therapeutic situation.

In another embodiment, the present invention provides a composition ofimmobilized or encapsulated cells, such as bacteria, or enzyme forlowering triglycerides. The phrase “triglyceride lowering amount” asused herein means an amount effective, at dosages and for periods oftime necessary to achieve the desired results (e.g. degradation oftriglyceride). Effective amounts may vary according to factors such asthe disease state, age, sex, weight of the animal. Dosage regima may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionately reduced as indicated by the exigencies of thetherapeutic situation.

In another embodiment, the present invention provides a compositioncomprising an immobilized and biologically active agent in an amountsufficient to degrade bile acids in association with a carrier. Thecomposition is optionally a pharmaceutical composition and the carrieris optionally a pharmaceutically acceptable carrier. The biologicallyactive agent may be any cell expressing a bile acid degrading enzyme,anaerobic bacteria expressing a bile acid degrading enzyme, a bile aciddegrading enzyme-containing cell extract or a bile acid degradingenzyme. Useful expressing cells include cells “capable of expressing”which means that the cell has an inducible element such that the enzymeis expressed when induced. “Bile acid degrading” means the ability tobreak down the conjugated bile acids glycodeoxycholic acid (GDCA) andtaurodeoxycholic acid (TDCA).

Cells or bacteria may be genetically engineered or produced by othermethods, such as irradiation-induced mutation or selection of naturallymutated cells that degrade increased amounts of bile acid compared to awild type cell. The cells are optionally any cell, such as an animalcell (eg. human cell) or a fungal cell or a bacterial cell as long asthey are capable of expressing the enzyme. The bacteria is optionallyLactobacillus such as, Lactobacillus plantarum, Lactobacillus reuteri ora combination thereof. The bacteria is optionally Bifidobacteriumbifidum, Lactobacillus acidophilus, or clostridium perfringens.

Suitable enzymes include various BSH enzymes and lipase. BSH isoptionally lactobacillus plantarum BSH and lipase is optionally animal(eg. mammalian, human) lipase. The L. plantarum BSH nucleotide sequenceis found in SEQ. ID. NO. 1 (Accession No. A24002) and the correspondingamino acid sequence is found in SEQ. ID. NO. 2 (Accession No. CAA01703).Alternatively, BSH is Bifidobacterium bifidum, Lactobacillusacidophilus, and Clostridium perfringens. The Bifidobacterium bifidumBSH nucleotide sequence is found in SEQ. ID. NO. 9 (Accession No.AY506536) and the corresponding amino acid sequence is found in SEQ. ID.NO. 10 (Accession No. AAR39453). The Lactobacillus acidophilus BSHnucleotide sequence is found in SEQ. ID. NO. 5 (Accession No. AF091248)and the corresponding amino acid sequence is found in SEQ. ID. NO. 6(Accession No. AAD03709). The clostridium perfringens BSH nucleotidesequence is found in SEQ. ID. NO. 7 (Accession No. U20191) and thecorresponding amino acid sequence is found in SEQ. ID. NO. 8 (AccessionNo. AAC43454). Enzyme is prepared by transcription and translation of anisolated nucleotide sequence or by de novo protein synthesis. Lipase isoptionally human lipase. The human lipase nucleotide sequence is foundin SEQ. ID. NO. 3 (Accession No. NM_(—)000235) and the correspondingamino acid sequence is found in SEQ. ID. NO. 4 (Accession No.NP_(—)000226).

Those skilled in the art will recognize that the enzyme nucleic acidmolecule sequences are not the only sequences, which may be used to makeproteins with enzymatic activity. The genetic code is degenerate soother nucleic acid molecules, which encode a polypeptide identical to anamino acid sequence of the present invention, may also be used. Thesequences of the other nucleic acid molecules of this invention may alsobe varied without changing the polypeptide encoded by the sequence.Consequently, the nucleic acid molecule sequences described below aremerely illustrative and are not intended to limit the scope of theinvention.

The sequences of the invention can be prepared according to numeroustechniques. The invention is not limited to any particular preparationmeans. For example, the nucleic acid molecules of the invention can beproduced by cDNA cloning, genomic cloning, cDNA synthesis, polymerasechain reaction (PCR), or a combination of these approaches (CurrentProtocols in Molecular Biology (F. M. Ausbel et al., 1989).). Sequencesmay be synthesized using well-known methods and equipment, such asautomated synthesizers.

The invention includes modified nucleic acid molecules with a sequenceidentity at least about: >17%, >20%, >30%, >40%, >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to a DNAsequence in SEQ. ID. NO. 1, 3, 5, 7 or 9 (or a partial sequencethereof). Preferably about 1, 2, 3, 4, 5, 6 to 10, 10 to 25, 26 to 50 or51 to 100, or 101 to 250 nucleotides or amino acids are modified.Identity is calculated according to methods known in the art. Sequenceidentity is most preferably assessed by the algorithm of the BLASTversion 2.1 program advanced search. Identity is calculated according tomethods known in the art. Sequence identity (nucleic acid and protein)is most preferably assessed by the algorithm of BLAST version 2.1advanced search. BLAST is a series of programs that are available onlineat http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast search(http://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=1) is set to defaultparameters. (ie Matrix BLOSUM62; Gap existence cost 11; Per residue gapcost 1; Lambda ratio 0.85 default). References to BLAST searches are:Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410;Gish, W. & States, D. J. (1993) “Identification of protein codingregions by database similarity search.” Nature Genet. 3:266272; Madden,T. L., Tatusov, R. L. & Zhang, J. (1996) “Applications of network BLASTserver” Meth. Enzymol. 266:131_(—)141; Altschul, S. F., Madden, T. L.,Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997)“Gapped BLAST and PSI_BLAST: a new generation of protein database searchprograms.” Nucleic Acids Res. 25:33893402; Zhang, J. & Madden, T. L.(1997) “PowerBLAST: A new network BLAST application for interactive orautomated sequence analysis and annotation.” Genome Res. 7:649656.

Nucleotide sequences functionally equivalent to BSH (SEQ. ID. NO. 1, 5,7 and 9) or lipase (SEQ. ID. NO. 3) can occur in a variety of forms asdescribed below. Polypeptides having sequence identity may be similarlyidentified.

The polypeptides encoded by the BSH or lipase nucleic acid molecules inother species will have amino acid sequence identity at leastabout: >20%, >25%, >28%, >30%, >40% or >50% to an amino acid sequenceshown in SEQ. ID. NO. 2, 4, 6, 8 or 10 (or a partial sequence thereof).Some species may have polypeptides with a sequence identity of at leastabout: >60%, >70%, >80% or >90%, more preferably at leastabout: >95%, >99% or >99.5% to all or part of an amino acid sequence inSEQ. ID. NO. 2, 4, 6, 8 or 10 (or a partial sequence thereof). Identityis calculated according to methods known in the art. Sequence identityis most preferably assessed by the BLAST version 2.1 program advancedsearch (parameters as above). Preferably about: 1, 2, 3, 4, 5, 6 to 10,10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides or aminoacids are modified.

The invention includes nucleic acid molecules with mutations that causean amino acid change in a portion of the polypeptide not involved inproviding bile acid degrading/triglyceride degrading activity or anamino acid change in a portion of the polypeptide involved in providingenzymatic activity so that the mutation increases or decreases theactivity of the polypeptide.

Other functional equivalent forms of the enzyme nucleic acid moleculesencoding nucleic acids can be isolated using conventional DNA-DNA orDNA-RNA hybridization techniques. These nucleic acid molecules and theenzyme sequences can be modified without significantly affecting theiractivity.

The present invention also includes nucleic acid molecules thathybridize to BSH or lipase sequences (or a partial sequence thereof) ortheir complementary sequences, and that encode peptides or polypeptidesexhibiting substantially equivalent activity as that of a BSH or lipasepolypeptide produced by the DNA in SEQ. ID. NO. 1, 3, 5, 7 or 9. Suchnucleic acid molecules preferably hybridize to all or a portion of thesequence or its complement under low, moderate (intermediate), or highstringency conditions as defined herein (see Sambrook et al. (mostrecent edition) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al.(eds.), 1995, Current Protocols in Molecular Biology, (John Wiley &Sons, NY)). The portion of the hybridizing nucleic acids is typically atleast 15 (e.g. 20, 25, 30 or 50) nucleotides in length. The hybridizingportion of the hybridizing nucleic acid is at least 80% e.g. at least95% or at least 98% identical to the sequence or a portion or all of anucleic acid encoding a BSH or lipase polypeptide, or its complement.Hybridizing nucleic acids of the type described herein can be used, forexample, as a cloning probe, a primer (e.g. a PCR primer) or adiagnostic probe. Hybridization of the oligonucleotide probe to anucleic acid sample typically is performed under stringent conditions.Nucleic acid duplex or hybrid stability is expressed as the meltingtemperature or Tm, which is the temperature at which a probe dissociatesfrom a target DNA. This melting temperature is used to define therequired stringency conditions. If sequences are to be identified thatare related and substantially identical to the probe, rather thanidentical, then it is useful to first establish the lowest temperatureat which only homologous hybridization occurs with a particularconcentration of salt (e.g. SSC or SSPE). Then, assuming that 1%mismatching results in a 1 degree Celsius decrease in the Tm, thetemperature of the final wash in the hybridization reaction is reducedaccordingly (for example, if sequences having greater than 95% identitywith the probe are sought, the final wash temperature is decreased by 5degrees Celsius). In practice, the change in Tm can be between 0.5degrees Celsius and 1.5 degrees Celsius per 1% mismatch. Low stringencyconditions involve hybridizing at about: 1×SSC, 0.1% SDS at 50° C. forabout 15 minutes. High stringency conditions are: 0.1×SSC, 0.1% SDS at65° C. for about 15 minutes. Moderate stringency is about 1×SSC 0.1% SDSat 60 degrees Celsius for about 15 minutes. The parameters of saltconcentration and temperature can be varied to achieve the optimal levelof identity between the probe and the target nucleic acid.

The present invention also includes nucleic acid molecules from anysource, whether modified or not, that hybridize to genomic DNA, cDNA, orsynthetic DNA molecules that encode the amino acid sequence of a BSH orlipase polypeptide, or genetically degenerate forms, under salt andtemperature conditions equivalent to those described in thisapplication, and that code for a peptide, or polypeptide that has bileacid degrading/triglyceride degrading activity. Preferably thepolypeptide has the same or similar activity as that of a BSH or lipasepolypeptide.

The invention also includes nucleic acid molecules and polypeptideshaving sequence similarity taking into account conservative amino acidsubstitutions. Changes in the nucleotide sequence which result inproduction of a chemically equivalent or chemically similar amino acidsequence are included within the scope of the invention. Variants of thepolypeptides of the invention may occur naturally, for example, bymutation, or may be made, for example, with polypeptide engineeringtechniques such as site directed mutagenesis, which are well known inthe art for substitution of amino acids. For example, a hydrophobicresidue, such as glycine can be substituted for another hydrophobicresidue such as alanine. An alanine residue may be substituted with amore hydrophobic residue such as leucine, valine or isoleucine. Anegatively charged amino acid such as aspartic acid may be substitutedfor glutamic acid. A positively charged amino acid such as lysine may besubstituted for another positively charged amino acid such as arginine.

Therefore, the invention includes polypeptides having conservativechanges or substitutions in amino acid sequences. Conservativesubstitutions insert one or more amino acids, which have similarchemical properties as the replaced amino acids. The invention includessequences where conservative substitutions are made that do not destroybile acid degrading/triglyceride degrading activity.

Polypeptides comprising one or more d-amino acids are contemplatedwithin the invention. Also contemplated are polypeptides where one ormore amino acids are acetylated at the N-terminus. Those of skill in theart recognize that a variety of techniques are available forconstructing polypeptide mimetics with the same or similar desired bileacid degrading activity as the corresponding polypeptide compound of theinvention but with more favorable activity than the polypeptide withrespect to solubility, stability, and/or susceptibility to hydrolysisand proteolysis. See, for example, Morgan and Gainor, Ann. Rep. Med.Chem., 24:243-252 (1989). Examples of polypeptide mimetics are describedin U.S. Pat. Nos. 5,643,873. Other patents describing how to make anduse mimetics include, for example in, 5,786,322, 5,767,075, 5,763,571,5,753,226, 5,683,983, 5,677,280, 5,672,584, 5,668,110, 5,654,276,5,643,873. Mimetics of the polypeptides of the invention may also bemade according to other techniques known in the art. For example, bytreating a polypeptide of the invention with an agent that chemicallyalters a side group by converting a hydrogen group to another group suchas a hydroxy or amino group. Mimetics preferably include sequences thatare either entirely made of amino acids or sequences that are hybridsincluding amino acids and modified amino acids or other organicmolecules.

The invention also includes polypeptide fragments of the polypeptides ofthe invention which may be used to confer bile acid degrading activityif the fragments retain activity. The invention also includespolypeptide fragments of the polypeptides of the invention which may beused as a research tool to characterize the polypeptide or its activity.Such polypeptides preferably consist of at least 5 amino acids. Inpreferred embodiments, they may consist of 6 to 10, 11 to 15, 16 to 25,26 to 50, 51 to 75, 76 to 100 or 101 to 250 amino acids of thepolypeptides of the invention (or longer amino acid sequences). Thefragments preferably have bile acid degrading/triglyceride degradingactivity.

Known techniques are used to bind enzyme or bacteria to a support, suchas a polymer bead. In short, the technique optionally involves simpleimmobilization/entrapment of the enzyme molecules in a support systemlike polymers. The support or bead may be made from solid or semi-solidmaterial. It may also be a porous support, hollow support or acontinuous support (without pores or hollows).

The immobilized biological active agent is optionally encapsulated ormicroencapsulated. Encapsulation is a term used to include the methodsof both macroencapsulation and microencapsulation. The termmicroencapsulation refers to a subclass of encapsulation, where small,microencapsulated capsules are produced. Encapsulation andmicroencapsulation techniques are known in the art. Microcapsules aresmall spherical containers or coated tissues in the 0.3-1.5 mm range,whereas macrocapsules are larger flat-sheet or hollow-fiber membranedvessels. Both macro- and microcapsules must contain a cellularenvironment that is able to support cellular metabolism andproliferation, as the cells they accommodate provide the capsulefunctionality.

Artificial cell microencapsulation is a technique used to encapsulatebiologically active materials in specialized ultra thin semi-permeablepolymer membranes (Chang and Prakash, 1997; Chang, 1964). Methods forpreparing artificial cells have been well documented in the pertinentart. Artificial cell membranes are optionally selected or designed foreach specific therapeutic device by one of skill in the art, because onemay engineer several different membranes for artificial cellpreparations with required membrane properties for a desiredapplication. The use of different membranes allows for variation inpermeability, mass transfer, mechanical stability, buffering capability,biocompatibility, and other characteristics. A balance has to bemaintained among the physical properties of capsule membranes so as tosupport the entrapped cell's survival.

The mass transport properties of a membrane are critical since theinflux rate of molecules, essential for cell survival, and the outflowrate of metabolic waste ultimately determines the viability of entrappedcells. Any barriers can be potentially applied to enzyme applications.Ordinarily the desired capsule permeability is determined by themolecular weight cut-off (MWCO), and is application dependent. The MWCOis the maximum molecular weight of a molecule that is allowed passagethrough the pores of the capsule membrane (Uludag et al. (2000) Adv.Drug Deliv. Rev. 42:29-64). For transplantation, the MWCO must be highenough to allow passage of nutrients, but low enough to rejectantibodies and other immune system molecules. The MWCO range isoptionally 3000 D to 950,000 D (Chang and Prakash, 1998). The MWCO oforally delivered microcapsules must allow for the passage of unwantedmetabolites from the plasma into the microcapsule, and then must eitherfacilitate the subsequent removal of the altered molecule or provide forits storage (Uludag et al., 2000). For cells of the invention that areto be administered orally or implanted in the gastrointestinal tract,one optionally uses a retainer that allows passage of nutrients, butblocks antibodies and other immune molecules, for example asemi-permeable membrane having a MWCO 3000 D to 950,000 D (Chang andPrakash, 1998). Alternatively, the lower end of the range may be about:2000D, 4000D, 5000D or 10,000D and the higher end of the range may beabout: 900,000D, 750,000D or 500,000D.

The most common type of membrane used for cell therapy is the singlealginate based polymer membrane; however, several other substances maybe used such as various proteins, polyhemoglobin, and lipids (Uludag etal., 2000; Prakash and Jones, 2002). Yet another approach for membranecomposition is to use a biodegradable synthetic polymer such aspolylactide, polyglycolic acid, and polyanhydride. Commonly usedmembranes include hollow fiber Membranes, alginate-polylysine-alginate(APA) membrane, cellulose nitrate, polyamide, lipid-complexed polymer,and lipid vesicles. Established and promising polymers for live cellencapsulation and enzyme encapsulation includealginate-polylysine-alginate (APA),alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),hydroxymethylacrylate-methyl methacrylate (HEMA-MMA), MultilayeredHEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC),acrylonitirle/sodium methallylsuflonate (AN-69), polyethyleneglycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane(PEG/PD₅/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), Siliceousencapsulates, and cellulose sulphate/sodiumalginate/polymethylene-co-guanidine (CS/A/PMCG) (with permission fromSatya Prakash and Hahn Soe-Lin, unpublished work). Other materials thatare useful include cellulose acetate phthalate, calcium alginate andk-carrageenan-Locust bean gum gel beads, gellan-xanthan beads,poly(lactide-co-glycolides), carageenan, starch polyanhydrides, starchpolymethacrylates, polyamino acids, enteric coating polymers.

The design of a membrane, intended for use in oral live cell therapy orenzyme therapy, must take into consideration several primary factors soas to minimize microbial death and maximize therapeutic effectiveness.To assure their efficacy, artificial cells intended for oraladministration, must be designed to protect their living cargo againstboth the acidic environment of the stomach and immunoglobulin releasedby the intestinal immune response.

A useful formulation is the encapsulation of calcium alginate beads withpoly-L-lysine (PLL) forming alginate-poly-L-lysine-alginate (APA)microcapsules. In the APA membrane microcapsule, alginate forms the coreand matrix for the cell and PLL binds to the alginate core. Binding ofPLL to alginate is the result of numerous long-chain alkyl-amino groupswithin PLL that extend from the polyamide backbone in a number ofdirections and interact with various alginate molecules, throughelectrostatic interactions. The resulting cross-linkage produces astable complex membrane that reduces the porosity of the alginatemembrane and forms an immunoprotective barrier.

Alternatively, Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate(APPPA), Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP), andAlginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA) membranesis used for encapsulation. It has been shown that these multi-layermembrane formulations perform well in GI stability tests, providing forincreased resistance to complete dissolution in water, dilute acids andbase, as well as in the presence of ion chelators, while allowing formore precise control over membrane permeability (Ouyang et al., 2002;Chen et al., 2002).

There are various methods available for preparing artificial cellscontaining live cells for therapy. For example, for preparation of theclassic alginate-polylysine-alginate (APA) membrane, the live cells,such as bacterial cells, are suspended in a matrix of the naturalpolymer alginate (1.5%). The viscous polymer-bacterial suspension ispassed through a 23-gauge needle using a syringe pump. Sterilecompressed air, passed through a 16-gauge coaxial needle, is then usedto shear the droplets coming out of the tip of the 23-gauge needle. Thedroplets are allowed to gel for 15 minutes in a gently stirred ice-coldsolution of solidifying chemicals, such as CaCl₂ (1.4%). After gelationin the CaCl₂, the beads are then washed with HEPES (0.05% in HEPES, pH7.20), coated with polylysine (0.1% for 10 min) and washed again inHEPES (0.05% in HEPES, pH 7.20). The resultant capsules are then coatedby reaction with alginate (0.1% for 10 min) and washed with appropriatechemicals to dissolve their inner core content. For this step a 3.00%citrate bath (3.00% in 1:1 HEPES-buffer saline, pH 7.20) is often used.The microcapsules formed can then be stored at 4° C. in minimal solution(10% cell nutrient to 90% water).

In one embodiment, the carrier is intended for oral administration andis optionally in the form of a nutraceutical or functional food product.“Nutraceutical” means a product isolated or purified from foods (orsources used to make food, such as plants, animals or other organisms)that is generally sold in a medicinal form not usually associated withfood. A nutraceutical is demonstrated to have a physiological benefit orprovide protection against chronic disease. “Functional Food Product”means it is similar in appearance to or may be a conventional food, isconsumed as part of a usual diet and is demonstrated to havephysiological benefits and/or reduce the risk of chronic disease beyondbasic nutritional functions.

In another embodiment, the carrier is an implantation device. Forexample, it is useful when administered to an appropriate place in theGI tract using implantable bags/pouches. Implantation of artificialcells has been described for the treatment of many disorders includinghepatic failure, pancreatic failure (Type I child onset diabetes), andalpha-1-antitrypsin deficiency (Moraga et al., 2001; Ambrosino et al.,2003). The procedure is common and known to one skilled in the art ofcell transplantation. To summarize, the capsules are inserted into theperitoneal cavity and interface with the visceral circulation. Thecapsules can then be retrieved.

Unwanted intraluminal bile acids in the gastrointestinal system isassociated with defective ileal transport of bile acids due to acongenital defect, resection of the ileum or bowel diseases. “Defectiveileal transport” means that there is excessive bile flow into the blood.Accordingly, the present invention provides a method for lowering ofintraluminal bile acid of patients suffering from defective ilealtransport of bile acids due to a congenital defect, resection of theileum or a bowel disease, which comprises of administering a bile acidlowering amount of a composition of the present invention.

The present inventors have demonstrated that microencapsulated cells,such as bacteria expressing BSH, can degrade bile salts. Accordingly,the present invention provides a method for lowering of serumcholesterol of an animal, which comprises administering a bile acidlowering amount of a composition of the present invention. In anotherembodiment, the invention provides a method for preventing or treatingany disease or disorder characterized by cholesterol or having excessivecholesterol as a risk factor comprising administering a bile acidlowering amount of a composition of the present invention. Cholesteroldisorders include familial hypercholesterolemia or inherited cholesteroldisorder (ICD), defects in the gene products of cholesterol metabolisme.g. 7-alpha-hydroxylase, and various forms of xanthomas. Increasedlevels of serum cholesterol may indicate atherosclerosis, biliarycirrhosis, familial hyperlipidemias, high-cholesterol diet,hypothyroidism, myocardial infarction, nephritic syndrome anduncontrolled diabetes. “Excessive cholesterol” means outside the optimalcholesterol range. Optimal cholesterol level is less than 200 mg/dL.Borderline High is 200-239 mg/dL and anything over 240 mg/dL is high.(http://www.nhlbi.nih.gov/helath/public/heart/chol/wyntk.htm). TheNational Cholesterol Education Program NCEP III report on cholesterol(http://www.nhlbi.nih.gov/guidelines/cholesterol/) includes “FullReport” and a “Drug Therapy” section. This provides a review of examplesof cholesterol management by statins, bile acid sequestrants, diet, etc.and it relates to cholesterol levels and risk factors (eg. see TablesIV.1-1 VI.1-1; VI.1-2, VI.1-3). The present invention drug is similar tobile acid sequestrants. The NCEP report provides guidance on use ofpharmaceutical therapy in relation to the presence of other riskfactors. There are two types of cholesterol, HDL cholesterol (sometimescalled good cholesterol) and LDL cholesterol (sometimes called badcholesterol). “Excessive cholesterol” may also be determined withrespect to LDL. For example, drug therapy is optionally considered forindividuals with multiple risk factors (2 or more) when LDL cholesterolis: >100 mg/dL (eg. with a goal to reduce LDL cholesterol to <100mg/dL), at least 130 mg/dL (eg. with a goal to reduce LDL cholesterol toless than 130 mg/dL), at least 160 mg/dL (eg. with a goal to reduce LDLcholesterol to less than 130 mg/dL). Furthermore, drug therapy is alsooptionally considered for individuals with 0-1 risk factors when LDLcholesterol is at least 190 mg/dL (eg. with a goal to reduce LDLcholesterol for less than 160 mg/dL). Normal values tend to increasewith age, and premenopausal women have somewhat lower levels than men ofthe same age.

The composition for lowering of intraluminal bile acids or serumcholesterol may be administered singly or in combination with anothercholesterol lowering therapeutic. Another cholesterol loweringtherapeutic includes BAS Cholestyramine resin (Locholest, Questran),Colesevelam (WelChol), Colestipol (Colestid), lovastatin (Mevacor),pravastatin (Pravachol), zocor (Zocor), fluvastatin (Lescol) andatorvastatin (Lipitor), probiotic formulations containing other livebacterial cells and neutraceuticals and natural cholesterol loweringproducts such as carbohydrates.

In another embodiment, the present invention provides a method forlowering of serum cholesterol and/or total body cholesterol of animalsfor the purpose of producing animal products of reduced cholesterolcontent, which comprises administering a bile acid lowering amount of acomposition of the present invention. Animal products optionally includecow, pig, or poultry meat or products such as eggs and milk.

Colon cancer has been linked to diet and the proposed mechanism is thata high fat diet leads to an increased secretion of primary bile saltsinto the small intestine. The increased biliary secretion leads to theformation of higher levels of deconjugated bile acids that may thenexert their cytotoxic and mutagenic effect on the gastrointestinalmucosa (Oumi and Yamamoto, 2000). It is these conjugated bile saltswhich have been incriminated in colonic carcinogenesis and thus a systemfor their removal would be a valuable tool for the prevention of coloncancer. Accordingly, the present invention provides a method for(preventing or treating) colon cancer in a patient, which comprisesadministering a bile acid lowering amount of a composition of thepresent invention.

In one embodiment, the present invention provides a method forquantitatively measuring bile acids or triglycerides.

Urinary levels of sulfated bile acids are known to be significantlyelevated in liver disease and hepatobiliary disease. Accordingly, thepresent invention provides an in vitro diagnostic tool for liver orhepatobiliary diseases in a patient, which comprises

a) support;

b) at least one biologically active agent immobilized onto said support;

wherein the immobilized agent allows detection and/or measurement ofbile acid degradation when contacted with a biological sample. Thebiologically active agent may be any cell expressing or capable ofexpressing a bile degrading enzyme, anaerobic bacteria expressing orcapable of expressing a bile degrading enzyme, a bile degradingenzyme-containing cell extract or a bile degrading enzyme. The supportoptionally comprises a support means. The present invention alsoprovides an in vitro diagnostic tool for steathorrea in a patient whichcomprises:

-   -   a) a support;    -   b) a biologically active agent immobilized onto said support;        wherein the immobilized agent allows detection and/or        measurement of triglyceride degradation when contacted with a        biological sample.

The biological sample is optionally urine. Alternatively, the biologicalsample is blood, feces or vomit. The detection of bile acid/triglyceridedegradation is based on an indicator that changes colour. Alternatively,the detection measures the quantity of bile acid/triglyceridedegradation. Bile degrading enzyme includes BSH. BSH is optionallylactobacillus plantarum BSH. The L. plantarum BSH nucleotide sequence isfound in SEQ. ID. NO. 1 (Genbank Accession No. A24002) and thecorresponding amino acid sequence is found in SEQ. ID. NO. 2 (GenbankAccession No. CAA01703). Alternatively, BSH is Bifidobacterium bifidum,Lactobacillus acidophilus, or Clostridium perfringens. TheBifidobacterium bifidum BSH nucleotide sequence is found in SEQ. ID. NO.9 (Genbank Accession No. AY506536) and the corresponding amino acidsequence is found in SEQ. ID. NO. 10 (Genbank Accession No. AAR39453).The Lactobacillus acidophilus BSH nucleotide sequence is found in SEQ.ID. NO. 5 (Genbank Accession No. AF091248) and the corresponding aminoacid sequence is found in SEQ. ID. NO. 6 (Genbank Accession No.AAD03709). The clostridium perfringens BSH nucleotide sequence is foundin SEQ. ID. NO. 7 (Genbank Accession No. U20191) and the correspondingamino acid sequence is found in SEQ. ID. NO. 8 (Genbank Accession No.AAC43454). Triglyceride degrading enzyme includes lipase. The humanlipase nucleotide sequence is found in SEQ. ID. NO. 3 (Accession No.NM_(—)000235) and the corresponding amino acid sequence is found in SEQ.ID. NO. 4 (Accession No. NP_(—)000226). Enzyme is prepared bytranscription and translation of an isolated nucleotide sequence or byde novo protein synthesis.

Cells or bacteria may be genetically engineered or produced by othermethods, such as irradiation-induced mutation or selection of naturallymutated cells that degrade increased amounts of bile acid/triglyceridecompared to a wild type cell. The cells are optionally any cell, such asan animal cell (eg. human cell) or a fungal cell or a bacterial cell aslong as they are capable of expressing the enzyme. The bacteria isoptionally Lactobacillus preferably, Lactobacillus plantarum,Lactobacillus reuteri or a combination thereof. The bacteria isoptionally Bifidobacterium bifidum, Lactobacillus acidophilus, andclostridium peifringens.

The liver is a complex organ with interdependent metabolic, excretory,and defense functions. No single or simple test assesses overall liverfunction; sensitivity and specificity are limited. Use of severalscreening tests improves the detection of hepatobiliary abnormalities,helps differentiate the basis for clinically suspected disease, anddetermines the severity of liver disease. Accordingly, in a furtherembodiment, the present invention provides an in vitro method formeasuring bile acid which comprises

(a) contacting a biological sample with a tool of the present invention,

(b) contacting a control sample with a tool of the present invention,

(c) comparing the amount of degradation of bile acid in (a) to (b),wherein a higher level of degradation product in (a) than (b) isindicative of a liver or hepatobiliary disease.

For example, the degradation product may be at least 10%, at least 30%,at least 50% or at least 100% higher. In one embodiment, the biologicalsample is urine. Alternatively, the biological sample is feces, vomit orblood. Elevated urinary bile acid levels can occur in infants andadults, for example, in hepatobiliary diseases including hepatitis A, B,C, G, Infantile obstructive cholangiopathy, Autoimmune hepatitis,Neonatal hepatitis, Infantile obstructive cholangiopathy, Progressivefamilial intrahepatic cholestasis (PFIC) and Intrahepatic cholestasis.Infants can also have bileary atresia as seen in the article byToshihiro Muraji et al. 2003. Elevated levels of sulfated bile acid(USBA) ranged from 9.1 to 341.0 μmol/L (92.3±141.1). USBA is notnormally seen in urine. Liver or hepatobiliary disease also includesflumanant hepatic failure, general liver disease, gallstone disease andprimary biliary cirrhosis.

The present invention also provides an in vitro method for measuringtriglyceride comprising:

a) contacting a biological sample with a tool of the present invention

b) contacting a control sample with a tool of the present invention

c) comparing the amount of degradation of triglyceride in (a) and (b)

wherein a higher level of degradation product in (a) than (b) isindicative of high triglyceride fat content.

The pharmaceutical compositions of the invention can be administered tohumans or animals by a variety of methods including, but not restrictedto topical administration, oral administration, aerosol administration,intratracheal instillation, intraperitoneal injection, injection intothe cerebrospinal fluid, and intravenous injection in methods of medicaltreatment involving bile acid degrading activity. Dosages to beadministered depend on patient needs, on the desired effect and on thechosen route of administration.

The pharmaceutical compositions can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions which can beadministered to patients, and such that an effective quantity of thecell or enzyme is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA).

The methods of combining the active molecules with the vehicles orcombining them with diluents is well known to those skilled in the art.The composition could include a targeting agent for the transport of theactive compound to specified sites within tissue.

The following non-limiting examples are illustrative of the presentinvention:

Materials and Methods: Bacterial Strains and Cell Growth and SelectionConditions

Any cell, such as a bacterial cell, may be engineered to overexpress BSHor lipase as described in the application and using known techniques.Two suitable bacterial strains used were the bile salt hydrolytic (BSH)isogenic Lactobacillus plantarum 80 (pCBHl) (LP80 (pCBHl)) strain(Christiaens et al., 1992) and the Lactobacillus reuteri (L. reuteri)strain (De Boever et al., 2000). Overproduction of the BSH enzyme inLP80 (pCBHl) was obtained as described by Christiaens et al., 1992. TheBSH overproducing LP80 (pCBHl) strain carried the multicopy plasmidpCBHl which carried the LP80 (pCBHl) chromosomal bsh gene and a markergene, the erythromycin resistance gene.

The Lactobacillus strains are optionally grown in MRS broth at 37° C. ina bench top incubator or in an anaerobic growth cabinet. For the growthof LP80 (pCBHl), the MRS broth was supplemented with 100/μg/mlerythromycin to select for bacteria overexpressing BSH (ie. carrying themulticopy plasmid pCBHl).

Immobilization of Cells—Lactobacillus plantarum 80 (pCBHl) and/orLactobacillus reuter

50 ml of 1.5% low viscosity alginate solution was prepared and filteredthrough a 0.22 μm filter into a sterile 60 ml syringe. LP80 (pCBHl) wasgrown at 37° C. in MRS broth and prepared as a concentratedmicroorganism suspension by re-suspension of microorganism in 10 ml ofsterilized physiologic solution. L. reuteri was grown at 37° C. in MRS10 broth and was added to the LP80 (pCBHl) concentrated microorganismsuspension. The 10 ml concentrated microorganism suspension was added tothe 50 ml low viscosity alginate solution and mixed well. Thealginate/microorganism mixture was immobilized, through a 300 μm nozzle,into a filtered solution of CaCl with an encapsulator. This procedure isoptionally performed in a biological containment hood to assuresterility. The immobilized cultures was stored in 1.0 L minimal solution(10% MRS and 90% Physiologic Solution) at 4° C.

Microencapsulation of Lactobacillus plantarum 80 (pCBHl) and/orLactobacillus reuteri

The microencapsulation procedure followed the same steps as theimmobilization procedure described above with the addition of thefollowing steps. The immobilized LP80 (pCBHl) and L. reuteri alginatebeads were washed in autoclaved physiological solution (8.5 NaCl/L),placed in a 1% solution of poly-L-lysine for 10 min., washed inphysiological solution, placed in 1% solution of low-viscosity alginatefor 10 min. and washed in physiological solution a final time. Thisprocedure was performed in a biological containment hood to assuresterility. The microencapsulated LP80 (pCBHl) (FIG. 1) and L. reuteriwas then stored in 1.0 L minimal solution (10% MRS and 90% PhysiologicSolution) at 4° C.

Microencapsulation and/or Immobilization of Bile Salt Hydrolase (BSH)Enzyme

The microencapsulation and immobilization procedures for the free BSHenzyme followed the same steps as outlined in the procedures describedabove with the following changes. The free enzyme was added to aphysiological solution or was simply added to the alginate preparationprior to bead formation. This procedure was performed in a biologicalcontainment hood to assure sterility. The microencapsulated enzyme wasthen stored in 1.0 L minimal solution (10% MRS and 90% PhysiologicSolution) at 4° C.

BSH Activity of Immobilized Lactobacillus plantarum 80 (pCBHl) AlginateBeads

To investigate the BSH activity of the immobilized BSH overproducingLP80 (pCBHl), batch experiments were performed. Five grams cell dryweight (CDW) of immobilized LP80 (pCBHl) was added to fresh MRS broth towhich 10.0 mM GDCA and 5.0 mM TDCA were added. Samples were taken atregular time intervals during the 24 h incubation to determine the bilesalt concentration in the reaction vessels.

BSH Activity of Lactobacillus plantarum 80 (pCBHl) Microcapsules

To investigate the BSH activity of the microencapsulated BSHoverproducing LP80 (pCBHl), batch experiments were performed. Five gramscell dry weight (CDW) of microencapsulated LP80 (pCBHl) was added tofresh MRS broth to which 10.0 mM GDCA and 5.0 mM TDCA were added.Samples were taken at regular time intervals during the 24 h incubationto determine the bile salt concentration in the reaction vessels.

Bile Salt Hydrolase Assay

A modification of the HPLC-procedure described by Scalia (1988) was usedto determine BSH activity. Traditionally, in vitro bile acidexperimentation has involved the use of HPLC to determine the quantityof various tauro- and glycol-bile acids in complex mixtures of addedbile acids in complex aqueous media (Scalia, 1988; Cantafora et al,1987; Coca et al., 1994). Methods to separate such mixtures haverequired a lengthy workup involving a (1:4; v:v) sample:isopropanolextraction followed by evaporation and resuspension steps (Scalia, 1988;Cantafora et al, 1987; Coca et al., 1994; De Smet et al., 1994). Whilethis method can produce, accurate results, the time consuming and laborintensive workup step of evaporation was eliminated, allowing for anefficient workup while preserving the quality of bile acid separationand quantification (Jones et al., 2003).

Analyses were performed on a reversed-phase C-18 column: LiChrosorb™ RP18, 5 μm, 250×4.6 mm from HiChrom™ (Novato, Calif., USA). The HPLCsystem was made up of two ProStar™ 210/215 solvent delivery modules, aProStar™ 320 UV/Vis Detector, a ProStar™ 410 AutoSampler™, and Star LCWorkstation Version 6.0 software was used. The solvents used wereHPLC-grade methanol (solvent A), and solvent B, which was acetate bufferprepared daily with 0.5 M sodium acetate, adjusted to pH 4.3 witho-phosphoric acid, and filtered through a 0.22 μm filter. An isocraticelution of 70 percent solvent A and 30 percent solvent B was used at aflow rate of 1.0 ml/min at room temperature. An injection loop of 20 μlwas used, and the detection occurred at 205 nm within 25 min afterinjection of the bile salt extract.

Quarter ml samples to be analyzed were acidified by the addition of 2.5μl of 6 N HCl to stop any further enzymatic activity. A modification ofthe extraction procedure described by Cantafora was used (Cantafora etal., 1987; Jones et al., 2003). From the 0.25 ml sample, bile salts wereextracted using a solution of methanol (1:1; v:v). GCA was added as aninternal standard at 4.0 mM. The samples were mixed vigorously for 10min and centrifuged at 1000 g for 15 min. The supernatant was thenfiltered through a 0.22 μm syringe driven HPLC-filter and the sampleswere analyzed directly after filtration.

Preparation of Alginate Beads Containing Immobilized GeneticallyEngineered Lactobacillus plantarum 80 (pCBHl) Cells

Alginate beads containing genetically engineered Lactobacillus plantarum80 (pCBHl) cells (FIG. 1) were prepared using the methods describedabove and were stored at 4° C. for use in experiments. Sterileconditions and procedures were strictly adhered to during the process ofmicroencapsulation.

Preparation of Artificial Cell Microcapsules Containing GeneticallyEngineered Lactobacillus plantarum 80 (pCBHl) Cells

Artificial cell microcapsules containing genetically engineeredLactobacillus plantarum 80 (pCBHl) cells (FIG. 2) were prepared usingthe methods described above and were stored at 4° C. for use inexperiments. Sterile conditions and procedures were strictly adhered toduring the process of microencapsulation.

Determination of Bile Acids by HPLC

To generate a calibration curve for quantifying the HPLC sample results,known quantities of GDCA and TDCA were added to MRS broth and 0.25 mLsamples were analyzed using the modified HPLC bile salt hydrolase assayoutlined above. FIG. 3 shows the calibration curves for GDCA and TDCAwith a 4.0 mM GCA internal standard and correlation factors of 0.987599and 0.991610 respectively. It is clear that this method allows for theaccurate identification and quantitative measurements of various bileacids (Jones et al, 2003).

Results: Example 1 BSH Activity of Alginate Beads Containing ImmobilizedLactobacillus plantarum 80 (pCBHl)

To show the BSH activity of alginate beads containing immobilized LP80(pCBHl), previously stored at 4° C., 5 g CDW of immobilized LP80 (pCBHl)was incubated in MRS broth supplemented with 10.0 mM GDCA and 5.0 mMTDCA. The concentration of bile acids was monitored by analyzing mediasamples at regular intervals over 24 hours. FIG. 4 shows superimposedHPLC chromatograms of bile acids in reaction media taken from one of theexperiments at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Decreasing peakareas of TDCA and GDCA bile acids indicate BSH activity of alginatebeads containing immobilized LP80 (pCBHl). The internal standard was GCAand was the first peak eluted.

Example 2 BSH Activity of Lactobacillus plantarum 80 (pCBHl)Microcapsules

To show the BSH activity of microencapsulated LP80, previously stored at4° C., and to show that microencapsulated LP80 (pCBHl) depletes highconcentrations of bile acids, 5 g of microencapsulated LP80 (pCBHl) wasincubated in MRS broth supplemented with 10.0 mM GDCA and 5.0 mM TDCA.The concentration of bile acids was monitored by analyzing media samplesat regular intervals over 12 hours. FIG. 5 shows superimposed HPLCchromatograms of bile acids in reaction media taken from one of theexperiments at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Decreasing peakareas of TDCA and GDCA bile acids indicate BSH activity of LP80 (pCBHl)microcapsules. The internal standard was GCA and was the first peakeluted.

The BSH activity of 0.25 g CDW of microencapsulated LP80 (pCBHl) and0.26 g CDW immobilized LP80 (pCBHl), both previously stored at 4° C.,was determined and is shown in Table 1. The BSH activity of 0.25 g CDWof microencapsulated LP80 (pCBHl) was calculated based on the depletionof 0.2 mmol of GDCA in a 4 h period, and the BSH activity towards TDCAwas based on the breakdown of 0.1 mmol of TDCA in a 5 h period. The BSHactivity of 0.26. g CDW of immobilized LP80 (pCBHl) was calculated basedon the depletion of 0.2 mmol of GDCA in a 5 h period, and the BSHactivity towards TDCA was based on the breakdown of 0.1 mmol of TDCA ina 6 h period. Also, these calculations were based on the in-vitrodepletion of bile acids with LP80 (pCBHl) in 5.0 g alginatemicrocapsules in a complex mixture of the bile acids.

TABLE 1 Bile salt hydrolayse (BSH) activity (μmol DCA/mg CDW/h) ofimmobilized and microencapsulated Lactobacillus plantarum 80 (pCBHl),previously stored at 4° C., towards glyco- and tauro- bile acids. BSHactivity (μmol DCA/mg CDW · h) towards Strain GDCA TDCA DCA (conjugated)Immobilized LP80 0.17 0.07 0.24 (pCBHl) Micro. LP80 (pCBHl) 0.19 0.080.27

FIG. 6 shows the BSH activity of immobilized LP80 (pCBHl) in alginate inan in-vitro bile acid experiment over a 12 h period. The concentrationof GDCA and TDCA bile acids are shown to decrease over time. It is clearfrom FIG. 6 that the BSH activity of immobilized LP80 (pCBHl) beganimmediately and depleted GDCA at a greater initial rate. While TDCA alsobegan to breakdown immediately, it did so at a slower rate than GDCA.The removal of GDCA, however, experienced concentration effects as itdepleted and thus the breakdown of GDCA slowed as the experimentprogressed.

FIG. 7 shows the BSH activity of LP80 (pCBHl) microcapsules in thein-vitro bile acid experiment over a 12 h period. The concentration ofGDCA and TDCA bile acids are shown to decrease over time. It is clearfrom FIG. 7 that the BSH activity of LP80 (pCBHl) began immediately anddepleted GDCA at a greater initial rate. While TDCA also began tobreakdown immediately, it did so at a slower rate than GDCA. The removalof GDCA, however, experienced concentration effects as it was depletedearly and thus the breakdown of GDCA slowed as the experiment progressedand the BSH activity towards TDCA increased.

To show the fate of the products of deconjugation, an experiment wasperformed using a calibration of increasing concentrations of TDCA,GDCA, and DCA. FIG. 10A shows superimposed HPLC chromatograms of samplesat 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Decreasing peak areas of TDCAand GDCA bile acids indicate BSH activity of microencapsulated LP80(pCBHl). These results were compared to earlier studies usingimmobilized beads containing LP80 (pCBHl) (FIG. 10B) Decreasing peakareas of TDCA and GDCA bile acids show BSH activity of alginate beadscontaining immobilized LP80 (pCBHl). The peak detected just before themeasured TDCA peak was diminished totally within 4 h and corresponds tothe calibration peak of DCA. The absence of a corresponding peak in theencapsulation results shows the clear advantage of using encapsulatedcells.

Example 3 Experimental Rat Model and In-Vivo Experimental Procedure

The in-vivo animal study employs young male Wistar rats and shows thesuitability of the microcapsule formulation for oral delivery of livegenetically engineered LP80 cells and the efficacy of such encapsulatedbacteria in lowering total cholesterol and improving the lipid profile.A standard procedure (Usman & Hosono, 2000) for making an elevated bloodserum cholesterol rat model by feeding a cholesterol-rich diet is used.Although some effective CHD rat models exist, a model involving manualelevation of blood serum levels provides greater flexibility incontrolling cholesterol.

For the in-vivo experimental protocol, 24 Wistar rats (Charles RiverLaboratories, USA), aged seven weeks and weighing 175-200 g atreception, are placed two per cage and fed Purina rat chow for 1 week inorder to acclimate them to the facility (sterile room with controlledtemperature (22-24° C.) and alternating light and dark cycles). Food andwater are provided ad libitum throughout the experiment. After achievingbaseline cholesterol and triglyceride values over a three week period,the rats are randomly split into two groups, a control group (8 rats fedPurina rat chow) and a high cholesterol (HC) test group (16 rats fedPurina rat chow supplemented with 10% corn oil and 1.5% (wt/wt)cholesterol). Upon stabilization of the increased serum cholesterollevels in the test group, the cholesterol fed rats are randomly splitinto two equal groups for the purpose of running a control group of 8rats fed a HC diet and empty microcapsules against a treatment group of8 rats fed a HC diet and microcapsules containing LP80 (pCBHl). For theexperiments, empty control microcapsules or microcapsules containing asuitable amount of log phase genetically engineered bacteria aresuspended in 0.8-1.0 ml sterile normal saline in a 5 ml syringe. Thefloating microcapsules are orally forced fed to the test rats twicedaily using curved 16G-3½ stainless steel gavage. Upon re-stabilizationof the decreased serum cholesterol levels among the treatment group, thegavage feeding is stopped. Throughout the experiment, weight gain ineach group is monitored weekly. Venous blood samples (500 uL) arecollected every 4th day (preceded by a 12-14 hour fast) in serumclotting activator tubes. The samples are centrifuged at 2000 g for 20minutes, and the supernatant serum is assayed for total cholesterol, HDLcholesterol and triglycerides using a Hitachi 911 clinical chemistryanalyzer. LDL cholesterol is determined by formula. Fresh fecal samplesare obtained on a weekly basis for analysis of excreted bile acids.Fecal bile acids are extracted by the method of van der Meer et al.(Usman & Hosono, 2000) and the supernatants are assayed enzymatically.

Example 4 Experimental Hamster Model and In-Vivo Experimental Procedure

The second animal model employs male golden Syrian hamsters to evaluatethe efficacy and safety of orally delivering microencapsulated livegenetically engineered LP80 cells. The hamster is well-established fordemonstrating cholesterol and bile acid metabolism that accuratelymimics the human condition (Spady et al, 1985; Spady et al, 1986; Spadyand Dietschy, 1988; Imray et al. 1992). In relation to animals ofsimilar size, the hamster is unique in that it contains plasmacholesterol ester transfer protein and LDL-receptor mediated activitiesat levels similar to humans (Ahn et al, 1994; Chen et al, 1996;Remillard et al., 2001; Trautwein, E. A., 1993) and its closely suitedlipoprotein profile is useful for studying the effects of diet andpharmaceutical products on lipoprotein metabolism (Bravo et al., 1994).Furthermore, the hamster requires only small increases in dietarycholesterol to induce elevations in plasma lipid and lipoproteincholesterol concentrations (Terpstra et al. 1991; Kowala, 1993). Inparticular, the Bio F₁B strain (BioBreeders USA) male golden Syrianhamster are employed because of its characteristic phenotype whichpromotes diet-induced hyperlipidemia and atherosclerotic lesionformation (Terpstra et al, 1991). When administered a diet of elevatedcholesterol and saturated fat, the Bio F₁B model shows increased serumcholesterol levels more significantly in the VLDL and LDL fraction, ascompared to the HDL fraction, making the hyperlipidemic Bio F₁B modelmore useful for mimicking the human situation than other strains(Trautwein et al., 1993; Kowala et al., 1991; Trautwein et al, 1993a).

For the in-vivo animal study, 24 male golden Syrian hamsters (strain BioF₁B, BioBreeders USA), aged 4 weeks and weighing ˜70 g at reception, areplaced two per cage and fed rodent chow for 1 week in order to acclimatethem to the facility (sterile room with controlled temperature (22-24°C.) and alternating light and dark cycles). Food and water are providedad libitum. After baseline cholesterol and triglyceride values are notedover a period of three weeks, the hamsters are divided into two groups,a control group (8 hamsters fed rodent chow) and a test (HC) group (16hamsters fed a nonpurified hypercholesterolemic diet consisting ofrodent chow supplemented with 3% corn oil and 0.5% (wt/wt) cholesterol).Previous studies have shown that a nonpurified diet, as compared to asemipurified diet, induces a lipoprotein profile similar to humans(Wilson et al. 1999; Krause et al, 1992). Once the increasing serumcholesterol levels in the test group stabilize, the cholesterol fedhamsters are randomly split into two equal groups for the purpose ofrunning a control group of 8 hamsters fed a HC diet and emptymicrocapsules, against a treatment group of 8 hamsters fed a HC diet andmicrocapsules containing LP80 (pCBHl) bacterial cells. For theexperiments, empty control microcapsules or microcapsules containing asuitable amount of log phase genetically engineered bacteria aresuspended in 0.8-1.0 ml sterile normal saline in a 5 ml syringe. Thefloating microcapsules are then orally forced fed to the test rats usingcurved 16G-3½ stainless steel gavage twice daily. Upon re-stabilizationof the decreased serum cholesterol levels among the treatment group, thegavage feeding is stopped. Throughout the experiment, weight gain ineach group is monitored weekly. Venous blood samples are collected every4th day (preceded by a 12-14 hour fast) in serum clotting activatortubes. The samples are centrifuged, and the supernatant serum is assayedfor total cholesterol, HDL cholesterol, and triglycerides using aHitachi 911 clinical chemistry analyzer. LDL cholesterol is computed byformula. Fresh fecal samples are obtained on a weekly basis for analysisof excreted bile acids. Fecal bile acids are extracted by the method ofvan der Meer et al. (1985) and the supernatants are assayedenzymatically.

Results: Baseline Period:

The results will show that the whole experimental group (EG) of animalswill have an adequately homogeneous baseline level of blood serumcholesterol (TC, HDL and LDL), and triglycerides (TG). The results willalso show that the EG will have homogeneous body weights, will eat anddrink similar amounts, and gain weight within normal limits etc.Finally, the results will show that the free DCA concentration will bemoderate in fecal samples of the EG group.

Cholesterol Feeding Period:

The results will show that the cholesterol fed group (HC) (⅔) of animalswill have high TC, HDL, LDL, TG, and will gain weight while the animalsremaining (⅓) in the EG will maintain their normal levels. The resultswill show that the HC group will eat slightly less food by weight butwill eat a normal or slightly elevated number of calories, as they areeating food containing more calories. The results will show that the HCgroup will have an elevated level of free DCA in fecal samples while theremaining EG group will maintain normal levels.

Therapeutic Period:

The results will show that

-   -   1. The animals receiving micro LP80 (pCBHl) therapy in the HC        group (½ the HC group) will achieve normalized levels of TC,        HDL, LDL, and possibly TG to some extent;    -   2. while the empty micro group (placebo) will maintain        previously high levels of TC, HDL, LDL, and TG;    -   3. and the outstanding EG will continue to show normal levels.

The results will show that free DCA levels may normalize in the treatedgroup, but will be directly dependent on the microcapsule stability,survivability, and of course extraction techniques used (experimentalconditions).

Normalization Period:

The results will show that when all animals are returned to the normaldiet and the treatment with micro. is stopped that the remaining levelsof the HC group, and in particular the placebo group, will normalize tothe group of EG animals that have remained a control thorough.

Example 5 Intraluminal Bile Acid Removal for Patients with CongenitalDisease, Bowel Resection or Disease of the Bowel

Patients need an effective and safe system for the removal of excessbile acids, because elevated intraluminal concentrations of deconjugatedbile acids in the colon normally result in an increased secretion ofelectrolytes and water causing diarrhea (Hofmann, 1999).Microencapsulated LP80 (pCBHl) and/or L. reuteri and/or BSH enzyme isorally administered to deconjugate, precipitate, and then bindconjugated bile acids within the microcapsules thus avoiding problemsassociated with excessive electrolyte and water secretion and theresulting diarrhea.

Example 6 Bioavailability of Deoxycholic Acid (DCA) and Addressing thePotential Concerns of Bile Acid Deconjugation by-Products

The BSH enzyme, overproduced by LP80 (pCBHl), releases glycine ortaurine from the conjugated bile salt steroid core and generatesdeconjugated primary bile salts, which are less water-soluble and areexcreted more easily via the faeces. This provides lowering of serumcholesterol with microencapsulated LP80 (pCBHl). As seen in FIGS. 10Aand 10D, microencapsulated LP80 (pCBHl) was able to deconjugate GDCA andTDCA completely within 4 h and 5 h respectively. In other experiments,immobilized LP80 (pCBHl) was able to effectively break down GDCA andTDCA bile acids within 5 h and 6 h respectively (FIG. 10B). However,with immobilization the deconjugation product, deoxycholic acid (DCA),was detected (FIG. 10B, 10C). This shows that microencapsulated cellsdiminished the bioavailability of BSH deconjugated bile acids totally(FIG. 10) (FIG. 9). The proposed mechanism in explanation, withoutwishing to be bound by a particular explanation, is that withmicroencapsulation (as opposed to immobilization or free bacteria) thereis decreased mass transfer of newly deconjugated DCA (by BSH from LP80(pCBHl)) and that the added concentration and interaction time withinthe microcapsule allows for the total precipitation of DCA by calciumions. The calcium may be produced by the immobilized bacteria or may bethat which is bound within the alginate matrix originally from the CaClpolymerizing solution.

This finding improves the therapeutic properties of microencapsulatedLP80 (pCBHl) in several ways. For example, it addresses concerns overthe production of large amounts of deconjugated bile salts and theirassociation with an increased risk of developing colon cancer. Also, ifbile salts are actually being deconjugated, precipitated, and then boundwithin the microcapsule, microencapsulated LP80 (pCBHl) may be capableof removing all bile acid from the GI lumen. LP80 is only one example ofa cell that overproduces BSH. The same result occurs with any cell thatexpresses BSH, any bacteria that expresses BSH, any cell extractcontaining BSH and with BSH enzyme itself. This effect contrastsprevious results, using free bacteria, where the authors predicted onlyan improved clearance (from 95% for conjugated to 60% for deconjugated)of bile acids from the ECH and not total clearance (De Smet et al.,1994) (FIG. 8). Further, elevated intraluminal concentrations ofdeconjugated bile acids in the colon, normally resulting in an increasedsecretion of electrolytes and water and causing diarrhea (Hofmann,1999), would cease to present difficulty, as the deconjugated bile acidswould be entirely precipitated and bound within the microcapsules andexcreted in the stool. Thus, the invention provides a method forreducing bile salts in an animal, comprising administering to the animala capsule comprising an encapsulated agent for deconjugating andprecipitating the bile salts to produce a bile salt derivative. Themethod further comprises binding the bile salt derivative to the capsulewherein the capsule and bile salt derivative are excreted by the animal

Another method for dealing with the unwanted DCA byproduct is byco-encapsulating a bacterium specially intended for the purpose. Recentstudies have shown that the adverse effects of deconjugated bile saltscan be counteracted by the addition of another naturally occurringresident of the gastrointestinal tract, L. reuteri (De Smet et al.,1994). It appears that the cell toxicity, normally exhibited bydeconjugated primary bile salts and the type produced by LP80 (pCBHl)BSH activity, is totally counteracted by the addition of L. reuteri (DeSmet et al, 1994). L. reuteri precipitates the deconjugated bile saltsand physically binds the bile salts making them less bioavailable. Thus,microencapsulated LP80 (pCBHl) holds yet another advantage overadministration of the free bacteria. That is that L. reuteri bacteria isadded to the LP80 (pCBHl) microcapsule so that the two bacteria worktogether, first deconjugating conjugated bile salts and thenprecipitating and binding deconjugated bile salts (FIG. 11). Thus, theinvention provides a method for reducing bile salts in an animal,comprising administering to the animal a capsule comprising:

(a) a bacteria that deconjugates bile salts and

(b) a second bacteria that precipitates and binds the deconjugated bilesalts.

In one embodiment, the first bacteria is L. plantarum and the secondbacteria is L. reuteri.

This system may also work to improve the therapeutic properties ofmicroencapsulated LP80 (pCBHl) in several ways. Firstly, by decreasingthe deconjugated bile salts bioavailability it addresses the concernsthat large amounts of deconjugated bile salts have been associated withan increased risk for colon cancer. Secondly, by deconjugating,precipitating and then binding the bile acids within the microcapsule,microencapsulated LP80 (pCBHl) totality removes bile acid from the ECH,not just improving the possibility of bile acid excretion (from 95% forconjugated to 60% for deconjugated). This provides total control of theECH in a noninvasive way (FIG. 8). Thirdly, elevated intraluminalconcentrations of deconjugated bile acids in the colon which wouldnormally result in an increased secretion of electrolytes and watercausing diarrhea is no longer be a problem because the deconjugated bileacids are precipitated and bound within the microcapsules by L. reuteri.Finally, it is important to note that microencapsulating LP80 (pCBHl)and L. reuteri together allows for their protection from the low pH andharsh environment of the stomach contents, it gives them closeproximity, and provides ideal conditions for this system of bile acidremoval.

Example 7 Combination Cholesterol Lowering Therapy

It is now well known that statins increase the risk of myopathy inpatients receiving large dosages and in patients with renal or hepaticimpairment, serious infections, hypothyroidism, or advanced age(Association, A. P. New Product Bulletin, 2002). In such patients, andin patients with an inadequate LDL lowering response to statins, it iswidely accepted that combination therapy with a bile acid sequestrant orniacin should be considered (Association, A. P. New Product Bulletin,2002; Brown et al, 2001; Gupta and Ito, 2002; Kashyap et al, 2002).Microencapsulated cells or bacteria such as LP80 (pCBHl) and/or L.reuteri, and/or free BSH enzyme are useful cholesterol lowering agentsfor use in combination therapy with statins and other lipid loweringtherapies.

Example 8 Preventative Therapy for Colon Cancer

It is believed that thirty percent of all colon cancer deaths can belinked to diet (Stone and Papas, 1997). One mechanism for this closeassociation is that a high fat diet leads to an increased secretion ofprimary bile salts into the small intestine, where the indigenousmicroflora deconjugates the primary bile acids. The increased biliarysecretion leads to the formation of higher levels of deconjugated bileacids that may then exert their cytotoxic and mutagenic effect on thegastrointestinal. mucosa (Oumi and Yamamoto, 2000). It is theseconjugated bile salts which have been incriminated in coloniccarcinogenesis and thus a system for their removal would be a valuabletool for the prevention of colon cancer. Treatment with a composition ofthe invention, such as microencapsulated L. reuteri and/ormicroencapsulated LP80 (pCBHl) and L. reuteri together, removes unwantedand potentially harmful deconjugated bile acids, such as DCA, andprovides a safe and effective means for patients and the public toprevent this deadly disease (FIG. 12).

Example 9 In Vitro Diagnostic Tool for Liver Function and HepatobiliaryDiseases

Urinary levels of sulfated bile acids are known to be significantlyelevated in liver disease and hepatobiliary disease (Back P., 1988).Several research groups have directed their efforts towards detection ofthese levels because urinary analysis is noninvasive, and urinary levelsof sulfated bile acids are thought to be useful as an index of liverfunction and an indicator of hepatobiliary disease (Kobayashi et al,2002).

A diagnostic strip containing immobilized beads and/or microcapsulescontaining cells expressing BSH such as LP80 (pCBHl) and/or L. reuteri,and/or the BSH enzyme itself as well as a colored detector molecule, isused as a novel noninvasive diagnostic tool for liver function andhepatobiliary diseases in urine (FIG. 13). BSH is overproduced by LP80(pCBHl) cells and hydrolyzes available conjugated bile acids. L. reuteriprecipitates and binds the produced deconjugated bile acids. The coloreddetector molecule reacts with either the deconjugated bile acid or thereleased amino acid groups from deconjugation and produces a perceptiblechange in color. This diagnostic strip may be readily adapted for usewith lipase to diagnose elevated triglyceride levels.

Example 10 Component of Integrated Bioartificial Liver

Incorporation of immobilized beads and/or microcapsules containing cellsexpressing BSH, such as Lactobacillus plantarum 80 (pCBHl) (LP80(pCBHl)) and/or Lactobacillus reuteri (L. reuteri) and/or BSH enzyme isuseful if incorporated into a bioartificial liver for the removal ofunwanted bile acids that build up during liver disease. It is well knownthat the bioartificial liver (BAL) must provide both synthetic anddetoxifying functions (Rozga et al, 1993; Schafer and Shaw, 1989)normally performed by the liver. Several groups have developed a BALconsisting of isolated porcine (Abouna et al., 1999; Morsiani et al.,1998) or bovine hepatocytes in a hollow-fiber bioreactor. Recently,researchers have focused on the use of BAL to support patients withfulminant hepatic failure (FHF), in which impaired liver function isassociated with pathologically elevated levels of bile acids. In thiscase an effective BAL requires the ability to remove and process asignificant quantity of deconjugated bile acid. Incorporation ofimmobilized beads and/or microcapsules containing LP80 (pCBHl) and/or L.reuteri is used for the removal of unwanted and pathologically highlevels of bile acids if incorporated into a bioartificial liver (FIG.14).

Example 11 Lipase Degradation of Triglyceride

Purified Lipase enzyme is microencapsulated/immobilized (Lipase, typeVII, from Candidida rugosa, containing lactose as an extender, fromSigma) in APA or other membrane using procedures as indicated above.Microencapsulation of any lipase is useful. A simple batch bioreactor(one-step in-vitro method) is used to incubate the microLipase withvarious concentrations of triglycerides (TG) at duodenal pH (7-4) forthe approximately 4 hour transit time and samples are removed at regularintervals for TG measurement. This procedure is optionally repeated in aSimulated Human Gastrointestinal Model (SHIME) whereby the low pH (1-2)environment of the stomach, correct transit times, neutralization, andthe normal human pancreatic enzymes are simulated. The samples are alsooptionally tested in spectrophotometor-type assay, for example, on aHitachi 911 clinical chemistry analyzer.

Reaction:

The addition of microLipase to the reaction flask provides an immediatedecrease in the triglyceride concentration and causes the release ofglycerol and free fatty acids. The results of this experiment show thatencapsulated lipase is useful for treating the condition (disorder)Steatorrhea which is most often associated with disease of the pancreasand small bowel.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

All publications, patents and patent applications, including thepriority application U.S. patent application No. 60/450,334 filed Feb.28, 2003, are herein incorporated by reference in their entirety to thesame extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

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1-25. (canceled)
 26. A method for lowering of intraluminal bile acid ofan animal comprising administering to the animal a bile acid loweringamount of a composition comprising (a) a bile-permeable microcapsulecomprising (i) a bile acid degrading enzyme or (ii) a cell expressing abile acid degrading enzyme; and (b) a carrier.
 27. A method for loweringof intraluminal bile acid of an animal comprising administering to theanimal a microcapsule comprising: (a) a bile acid lowering amount of afirst bacteria that degrades bile salts and (b) a second bacteria thatprecipitates and binds the degraded bile salts.
 28. (canceled)
 29. Themethod of claim 26, wherein the composition lowers serum cholesterol ofthe animal.
 30. (canceled)
 31. The method of claim 29, wherein thecomposition is administered in combination with another cholesterollowering therapeutic.
 32. The method of claim 31, wherein the anothercholesterol lowering therapeutic is selected from the group consistingof BAS Cholestyramine resin, Colesevelam, Colestipol, statin, probioticformulation containing other live bacterial cells, neutraceuticals andnatural cholesterol lowering products.
 33. The method of claim 32wherein the statin is selected from the group consisting of lovastatin,pravastatin, simvastatin, fluvastatin and atorvastatin. 34-78.(canceled)
 79. The method of claim 29, wherein the serum cholesterol islow density lipoprotein cholesterol (LDL-C).
 80. The method of claim 79,wherein the LDL-C is lowered to a serum concentration below 130 mg/dl.81. The method of claim 26, wherein the composition lowers serumtriglycerides and/or body fat of the animal.
 82. The method of claim 26,wherein the bile acid degrading enzyme is present in an amountsufficient to decrease absorption of triglycerides in the intestinaltract, lower serum triglycerides, and/or lower body fat.
 83. The methodof claim 26, wherein the bile acid degrading enzyme is present in anamount sufficient to degrade bile in the small intestine.
 84. The methodof claim 26, wherein the bile acid degrading enzyme is present in anamount sufficient to lower serum cholesterol.
 85. The method of claim29, for treating a cholesterol disease or disorder or a disease ordisorder having cholesterol as a risk factor.
 86. The method of claim85, wherein the cholesterol disease or disorder is familialhypercholesterolemia, inherited cholesterol disorder, a defect in a geneproduct of cholesterol metabolism, or a xanthoma.
 87. The method ofclaim 85, wherein the disease or disorder having cholesterol as a riskfactor is atherosclerosis, biliary cirrhosis, familial hyperlipidemia,hypothyroidism, myocardial infarction, nephritic syndrome or diabetes.88. The method of claim 85, wherein the disease or disorder havingcholesterol as a risk factor is atherosclerosis.
 89. The method of claim85, wherein the disease or disorder having cholesterol as a risk factoris diabetes.
 90. The method of claim 26, wherein the microcapsulereduces exposure of the cell or enzyme to antibodies compared to a freecell or enzyme, but permits exposure to nutrients.
 91. The method ofclaim 26, wherein the microcapsule comprises a polymer bead and theenzyme or cell is immobilized in the bead.
 92. The method of claim 26,wherein the enzyme degrades bile acid to a target-degradation compound.93. The method of claim 92, wherein the target-degradation compoundcomprises deoxycholic acid (DCA) or a cholic acid precipitate.
 94. Themethod of claim 92, wherein the microcapsule or cell retains thetarget-degradation compound.
 95. The method of claim 26, wherein thecell is a human cell, a fungal cell or a bacterial cell.
 96. The methodof claim 95, wherein the bacterial cell is an anaerobic bacterial cell.97. The method of claim 95, wherein the cell is genetically engineered.98. The method of claim 95, wherein the bacterial cell is Lactobacillusor Bifidobacteria.
 99. The method of claim 95, wherein the bacterialcell is Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacteriumbifidum, Lactobacillus acidophilus or Clostridium perfringens.
 100. Themethod of claim 99, comprising Lactobacillus reuteri.
 101. The method ofclaim 26, wherein the bile acid degrading enzyme is bile salt hydrolase(BSH).
 102. The method of claim 101, wherein the BSH is encoded by anucleotide sequence as shown in one of SEQ ID NO: 1, 5, 7 or 9 orcomprises an amino acid sequence as shown in one of SEQ ID NO:2, 6, 8 or10.
 103. The method of claim 26, wherein the microcapsule comprises asynthetic polymer.
 104. The method of claim 103, wherein the syntheticpolymer comprises polylactide, polyglycolic acid or polyanhydride. 105.The method of claim 26, wherein the microcapsule comprises alginate.106. The method of claim 26, wherein the microcapsule comprisesalginate-polylysine-alginate (APA).
 107. The method of claim 26, whereinthe microcapsule comprisesAlginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP),Alginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA), oralginate-polymethylene-co-guanidine-alginate (A-PMCG-A).
 108. The methodof claim 26, wherein the microcapsule comprisesAlginate/Poly-l-lysine/Pectin/Poly-l-lysine/Alginate (APPPA),Alginate/Poly-l-lysine/Pectin/Poly-l-lysine/Pectin (APPPP),Alginate/Poly-L-lysine/Chitosan/Poly-l-lysine/Alginate (APCPA),alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),hydroxymethylacrylate-methyl methacrylate (HEMA-MMA), MultilayeredHEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC),acrylonitirle/sodium methallylsuflonate (AN-69), polyethyleneglycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane(PEG/PD₅/PDMS) or poly N,N-dimethyl acrylamide (PDMAAm) membranes. 109.The method of claim 26, wherein the microcapsule comprises hollow fiber,cellulose nitrate, polyamide, lipid-complexed polymer, a lipid vesicle,a siliceous encapsulate, cellulose sulphate/sodiumalginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetatephthalate, calcium alginate, k-carrageenan-Locust bean gum gel beads,gellan-xanthan beads, poly(lactide-co-glycolides), carageenan, starchpolyanhydrides, starch polymethacrylates, polyamino acids or entericcoating polymers.
 110. The method of claim 26, wherein thebile-permeable microcapsule has a molecular weight cutoff point (MWCO)of 3000 D to 950,000 D.
 111. The method of claim 26, wherein the carriercomprises an orally administrable carrier.
 112. The method of claim 26,wherein the carrier comprises a nutraceutical or functional foodproduct.
 113. The method of claim 26, wherein the carrier isimplantable.
 114. The method of claim 26, wherein the composition is apharmaceutical composition and the carrier is a pharmaceuticallyacceptable carrier.
 115. The method of claim 114, wherein thepharmaceutical composition further comprises an additional cholesterollowering therapeutic.
 116. The method of claim 115, wherein theadditional cholesterol lowering therapeutic is selected form the groupconsisting of bile acid sequesterant (BAS) Cholestyramine resin,Colesevelam, Colestipol, statin, a probiotic formulation containingother live bacterial cells, neutraceuticals and natural cholesterollowering products.
 117. The method of claim 116, wherein the statin isselected from the group consisting of lovastatin, pravastatin,simvastatin, fluvastatin and atorvastatin.
 118. The method of claim 26,wherein the animal is a human.